Integrated device with external light source for probing detecting and analyzing molecules

ABSTRACT

System and methods for analyzing single molecules and performing nucleic acid sequencing. An integrated device includes multiple pixels with sample wells configured to receive a sample, which when excited, emits radiation. The integrated device includes at least one waveguide configured to propagate excitation energy to the sample wells from a region of the integrated device configured to couple with an excitation energy source. A pixel may also include at least one element for directing the emission energy towards a sensor within the pixel. The system also includes an instrument that interfaces with the integrated device. The instrument may include an excitation energy source for providing excitation energy to the integrated device by coupling to an excitation energy coupling region of the integrated device. One of multiple markers distinguishable by temporal parameters of the emission energy may label the sample and configuration of the sensor within a pixel may allow for detection of a temporal parameter associated with the marker labeling the sample.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/845,996, titled “INTEGRATED DEVICE WITH EXTERNAL LIGHT SOURCE FORPROBING DETECTING AND ANALYZING MOLECULES,” filed Dec. 18, 2017, whichis a continuation of U.S. patent application Ser. No. 14/821,688, titled“INTEGRATED DEVICE WITH EXTERNAL LIGHT SOURCE FOR PROBING DETECTING ANDANALYZING MOLECULES,” filed Aug. 7, 2015, which claims priority to U.S.Provisional Patent Application No. 62/035,258, titled “INTEGRATED DEVICEWITH EXTERNAL LIGHT SOURCE FOR PROBING, DETECTING, AND ANALYZINGMOLECULES,” filed Aug. 8, 2014, and U.S. Provisional Patent ApplicationNo. 62/164,464, titled “INTEGRATED DEVICE WITH EXTERNAL LIGHT SOURCE FORPROBING, DETECTING, AND ANALYZING MOLECULES,” filed May 20, 2015, eachof which is hereby incorporated by reference in its entirety.

This application is related to the following U.S. applications:

-   -   U.S. Provisional Patent Application 62/164,506, titled        “INTEGRATED DEVICE FOR TEMPORAL BINNING OF RECEIVED PHOTONS”,        filed May 20, 2015;    -   U.S. Provisional Patent Application 62/164,485, titled “PULSED        LASER,” filed May 20, 2015;    -   U.S. Provisional Patent Application 62/164,482, titled “METHODS        FOR NUCLEIC ACID SEQUENCING,” filed May 20, 2015;    -   U.S. Provisional Patent Application 62/035,242, titled “OPTICAL        SYSTEM AND ASSAY CHIP FOR PROBING, DETECTING AND ANALYZING        MOLECULES,” filed Aug. 8, 2014, which is hereby incorporated by        reference in its entirety;    -   U.S. non-provisional patent application Ser. No. 14/821,656,        titled “INTEGRATED DEVICE FOR TEMPORAL BINNING OF RECEIVED        PHOTONS,” filed Aug. 7, 2015; and    -   U.S. non-provisional patent application Ser. No. 14/821,686,        titled “OPTICAL SYSTEM AND ASSAY CHIP FOR PROBING, DETECTING AND        ANALYZING MOLECULES,” filed Aug. 7, 2015.

Each of the above-listed related applications is hereby incorporated byreference in its entirety.

FIELD OF THE APPLICATION

The present application is directed generally to devices, methods andtechniques for performing rapid, massively parallel, quantitativeanalysis of biological and/or chemical samples, and methods offabricating said devices.

BACKGROUND

Detection and analysis of biological samples may be performed usingbiological assays (“bioassays”). Bioassays conventionally involve large,expensive laboratory equipment requiring research scientists trained tooperate the equipment and perform the bioassays. Moreover, bioassays areconventionally performed in bulk such that a large amount of aparticular type of sample is necessary for detection and quantitation.

Some bioassays are performed by tagging samples with luminescent markersthat emit light of a particular wavelength. The markers are illuminatedwith a light source to cause luminescence, and the luminescent light isdetected with a photodetector to quantify the amount of luminescentlight emitted by the markers. Bioassays using luminescent markersconventionally involve expensive laser light sources to illuminatesamples and complicated luminescent detection optics and electronics tocollect the luminescence from the illuminated samples.

SUMMARY

The technology described herein relates to apparatus and methods foranalyzing specimens rapidly using an active-source-pixel, integrateddevice that can be interfaced with a mobile computing instrument. Theintegrated device may be in the form of a disposable or recyclablelab-on-chip or a packaged module that is configured to receive a smallamount of a specimen and execute, in parallel, a large number ofanalyses of samples within the specimen. The integrated device may beused to detect the presence of particular chemical or biologicalanalytes in some embodiments, to evaluate a chemical or biologicalreactions in some embodiments, and to determine genetic sequences insome embodiments. According to some implementations, the integrateddevice may be used for single-molecule gene sequencing.

According to some implementations, a user deposits a specimen in achamber on the integrated device, and inserts the integrated device intoa receiving instrument. The receiving instrument, alone or incommunication with a computer, automatically interfaces with theintegrated device, receives data from the integrated device, processesthe received data, and provides results of the analysis to the user. Asmay be appreciated, integration and computing intelligence on the chip,receiving instrument, and or computer reduce the skill level requiredfrom the user.

According to some embodiments of the present application, an integrateddevice is provided, comprising a plurality of pixels. A pixel of theplurality of pixels comprises a sample well configured to receiveexcitation energy from an excitation source external to the integrateddevice and at least one sensor positioned to receive luminescence from asample positioned in the sample well and generate a signal that providesidentification information of the sample based on the receivedluminescence.

In some embodiments, the signal is indicative of a temporal parameter ofthe received luminescence. In some embodiments, the temporal parameteris a lifetime associated with the luminescence from the sample. In someembodiments, the signal is indicative of a spectrum of the luminescence.In some embodiments, the signal is indicative of a characteristicwavelength of the luminescence. In some embodiments, the signal and theexcitation energy indicates an absorption spectrum of the sample. Insome embodiments, the signal and the excitation energy indicates acharacteristic wavelength absorbed by the sample.

According to some embodiments of the present application, an integrateddevice is provided, comprising a pixel region comprising a plurality ofpixels. A pixel of the plurality of pixels has a sample well on asurface of the integrated device, wherein the sample well is configuredto receive a sample, at least one sensor configured to receiveluminescence from the sample well, and at least one waveguide fordelivering excitation energy to a vicinity of the sample well. Theintegrated device comprises an excitation source coupling regioncomprising a coupling component configured to receive excitation energyfrom an external excitation energy source and couple the excitationenergy into the waveguide.

According to some embodiments of the present application, a system isprovided, comprising an excitation source module comprising anexcitation source configured to emit a pulse of excitation energy havinga first duration of time and an integrated device. The integrated devicea sample well configured to receive a sample which, when coupled to thepulse of excitation energy emits luminescence, a sensor that detects theluminescence over a second duration of time, wherein the second durationof time occurs after the first duration of time, a first energy pathalong which the pulse of excitation energy moves from the excitationsource to an energy source coupling component, a second energy pathalong which the pulse of excitation energy moves from the energy sourcecoupling component to the sample well, and a third energy path alongwhich the luminescence moves from the sample well to the sensor.

According to some embodiments of the present application, a method ofdetecting the presence of a molecule in a sample is provided. The methodcomprises introducing a sample labeled with one of a plurality ofluminescent markers into a sample well, wherein at least a portion ofthe plurality of luminescent markers having a different luminescentlifetime values. The method further comprises irradiating the samplewell with a pulse of light, measuring the time of arrival of photonsemitted from sample well, and determining the identity of a marker basedon the time of arrival of the photons.

According to some embodiments of the present application, an integrateddevice comprising a sample well and a sensor is provided. The samplewell is configured to receive a sample labeled with one of a pluralityof luminescent markers, each of the plurality of luminescent markers hasa different luminescent lifetime value. The sensor is configured todetect luminescence from one of the plurality of luminescent markersover a plurality of time durations, wherein the plurality of timedurations is selected to differentiate among the plurality ofluminescent markers.

According to some embodiments of the present application, an integrateddevice comprising a sample well and a plurality of sensors is provided.The sample well is configured to receive a sample labeled with one of aplurality of luminescent markers. Each of the plurality of luminescentmarkers emit luminescence within one of a plurality of spectral rangesand a portion of the plurality luminescent markers that emitluminescence at one of the plurality of spectral ranges each havedifferent luminescent lifetime values. Each sensor of the plurality ofsensors is configured to detect one of the plurality of spectral rangesover a plurality of time durations and the plurality of time durationsare selected to differentiate among the portion of the plurality ofluminescent markers.

According to some embodiments, a system comprising a plurality ofexcitation sources and an integrated device is provided. The pluralityof excitation sources is configured to emit a plurality of excitationenergies, wherein each of the plurality of excitation sources emitspulses of one of the plurality of excitation energies. The integrateddevice includes a sample well configured to receive a sample labeledwith one of a plurality of luminescent markers. A portion of theplurality of luminescent markers emit luminescence after beingilluminated by one of the plurality of excitation energies each havedifferent lifetime values. The integrated device further includes asensor configured to detect luminescence from one of the plurality ofluminescent markers over a plurality of time durations after a pulse ofone of the plurality of excitation energies, wherein a timing of thepulse of one of the plurality of excitation energies and the pluralityof time durations differentiate among the plurality of luminescentmarkers.

According to some embodiments of the present application, a method offorming an integrated device is provided. The method comprises forming aplurality of sensor regions, wherein a sensor region of the plurality ofsensor regions includes a plurality of sensors, forming a plurality ofsample wells, wherein a sample well of the plurality of sample wellsaligns with a corresponding one of the plurality of sensor regions, andforming at least one waveguide configured to couple excitation energyseparate from the plurality of sample wells and direct excitation energyto at least one sample well.

According to some embodiments of the present application, an instrumentis provided. The instrument comprises at least one excitation source forproviding at least one excitation energy, an excitation sourcepositioning system for aligning the at least one excitation energyemitted by the at least one excitation source to a coupling region of anintegrated device, and readout circuitry configured to receive at leastone readout signal representative of emission energy detected by asensor on the integrated device.

According to some embodiments of the present application, a method forsequencing a target nucleic acid molecule is provided. The methodcomprises providing an integrated device that includes a sample wellcontaining the target nucleic acid molecule, a polymerizing enzyme, anda plurality of types of nucleotides or nucleotide analogs. Each type ofnucleotide or nucleotide analog of the plurality of types of nucleotidesor nucleotide analogs is labeled with one or a plurality of markers. Themethod further comprises performing an extension reaction at a priminglocation of the target nucleic acid molecule in the presence of apolymerizing enzyme to sequentially incorporate at least a portion ofthe nucleotides or nucleotide analogs into a growing strand that iscomplementary to the target nucleic acid molecule, wherein uponexcitation by excitation energy the markers labelling the nucleotides ornucleotide analogs produce emissions from the sample well uponincorporation into the growing strand and emission lifetimes aredistinguishable for the plurality of types of nucleotides or nucleotideanalogs. The method further comprises identifying the nucleotides ornucleotide analogs based on signals received from the sensor that areindicative of the emission lifetimes, thereby sequencing the targetnucleic acid molecule.

According to some embodiments of the present application, a method fornucleic acid sequencing is provided. The method comprises providing anintegrated device comprising a plurality of sample wells and anexcitation energy source that is operatively coupled to the plurality ofsample wells. Am individual sample well of the plurality of sample wellscomprises a target nucleic molecule, a polymerizing enzyme andnucleotides or nucleotide analogs. One marker of a plurality of markerslabels each of the nucleotides or nucleotide analogs. The method furthercomprises subjecting the target nucleic acid molecule to apolymerization reaction to yield a growing strand that is complementaryto the target nucleic acid molecule in the presence of the nucleotidesor nucleotide analogs and the polymerizing enzyme. The plurality ofmarkers emits emissions upon excitation by excitation energy from theexcitation source while the nucleotides or nucleotide analogs areincorporated into the growing strand. The method further comprisesdetecting lifetimes of the emissions while performing the extensionreaction, wherein the lifetimes of the emissions are distinguishable forthe plurality of markers, and identifying a sequence of the targetnucleic acid molecule based on the lifetimes of the emissions.

According to some embodiments of the present application, a method ofanalyzing a specimen is provided. The method comprises depositing thespecimen on a surface of an integrated device having a plurality ofpixels, wherein each pixel has a sample well configured to receive asample labeled with a first marker of a plurality of markers and asensor region having at least one sensor, aligning the integrated devicewith an instrument having at least one excitation energy source forcoupling excitation energy to a sample well of a first pixel and readoutcircuitry for receiving readout signals from the at least one sensor ofthe sensor region of the first pixel, illuminating the first marker withexcitation energy, and detecting, from the readout signals from the atleast one sensor of the sensor region of the first pixel, a lifetime ofthe emission energy generated from an emission by the first marker.

The term “pixel” may be used in the present disclosure to refer to aunit cell of an integrated device. The unit cell may include a samplewell and a sensor. The unit cell may further include at least oneexcitation-coupling optical structure (which may be referred to as a“first structure”) that is configured to enhance coupling of excitationenergy from the excitation source to the sample well. The unit cell mayfurther include at least one emission-coupling structure that isconfigured to enhance coupling of emission from the sample well to thesensor. The unit cell may further include integrated electronic devices(e.g., CMOS devices). There may be a plurality of pixels arranged in anarray on an integrated device.

The term “optical” may be used in the present disclosure to refer tovisible, near infrared, and short-wavelength infrared spectral bands.

The term “tag” may be used in the present disclosure to refer to a tag,probe, or reporter and include a marker attached to a sample to beanalyzed or a marker attached to a reactant that may bind with a sample.

The phrase “excitation energy” may be used in the present disclosure torefer to any form of energy (e.g., radiative or non-radiative) deliveredto a sample and/or marker within the sample well. Radiative excitationenergy may comprise optical radiation at one or more characteristicwavelengths.

The phrase “characteristic wavelength” may be used in the presentdisclosure to refer to a central or predominant wavelength within alimited bandwidth of radiation. In some cases, it may refer to a peakwavelength of a bandwidth of radiation. Examples of characteristicwavelengths of fluorophores are 563 nm, 595 nm, 662 nm, and 687 nm.

The phrase “characteristic energy” may be used in the present disclosureto refer to an energy associated with a characteristic wavelength.

The term “emission” may be used in the present disclosure to refer toemission from a marker and/or sample. This may include radiativeemission (e.g., optical emission) or non-radiative energy transfer(e.g., Dexter energy transfer or Förster resonant energy transfer).Emission results from excitation of a sample and/or marker within thesample well.

The phrase “emission from a sample well” or “emission from a sample” maybe used in the present disclosure to refer to emission from a markerand/or sample within a sample well.

The term “self-aligned” may be used in the present disclosure to referto a microfabrication process in which at least two distinct elements(e.g., a sample well and an emission-coupling structure, a sample welland an excitation-source) may be fabricated and aligned to each otherwithout using two separate lithographic patterning steps in which afirst lithographic patterning step (e.g., photolithography, ion-beamlithography, EUV lithography) prints a pattern of a first element and asecond lithographic patterning step is aligned to the first lithographicpatterning step and prints a pattern of the second element. Aself-aligned process may comprise including the pattern of both thefirst and second element in a single lithographic patterning step, ormay comprise forming the second element using features of a fabricatedstructure of the first element.

The term “sensor” may be used in the present disclosure to refer to oneor more integrated circuit devices configured to sense emission from thesample well and produce at least one electrical signal representative ofthe sensed emission.

The term “nano-scale” may be used in the present disclosure to refer toa structure having at least one dimension or minimum feature size on theorder of 150 nanometers (nm) or less, but not greater than approximately500 nm.

The term “micro-scale” may be used in the present disclosure to refer toa structure having at least one dimension or minimum feature sizebetween approximately 500 nm and approximately 100 microns.

The phrase “enhance excitation energy” may be used in the presentdisclosure to refer to increasing an intensity of excitation energy atan excitation region of a sample well. The intensity may be increased byconcentrating and/or resonating excitation energy incident on the samplewell, for example. In some cases, the intensity may be increased byanti-reflective coatings or lossy layers that allow the excitationenergy to penetrate further into the excitation region of a sample well.An enhancement of excitation energy may be a comparative reference to anembodiment that does not include structures to enhance the excitationenergy at an excitation region of a sample well.

The terms “about,” “approximately,” and “substantially” may be used inthe present disclosure to refer to a value, and are intended toencompass the referenced value plus and minus acceptable variations. Theamount of variation could be less than 5% in some embodiments, less than10% in some embodiments, and yet less than 20% in some embodiments. Inembodiments where an apparatus may function properly over a large rangeof values, e.g., a range including one or more orders of magnitude, theamount of variation could be a factor of two. For example, if anapparatus functions properly for a value ranging from 20 to 350,“approximately 80” may encompass values between 40 and 160.

The term “adjacent” may be used in the present disclosure to refer totwo elements arranged within close proximity to one another (e.g.,within a distance that is less than about one-fifth of a transverse orvertical dimension of a pixel). In some cases there may be interveningstructures or layers between adjacent elements. In some cases adjacentelements may be immediately adjacent to one another with no interveningstructures or elements.

The term “detect” may be used in the present disclosure to refer toreceiving an emission at a sensor from a sample well and producing atleast one electrical signal representative of or associated with theemission. The term “detect” may also be used in the present disclosureto refer to determining the presence of, or identifying a property of, aparticular sample or marker in the sample well based upon emission fromthe sample well.

The foregoing and other aspects, embodiments, and features of thepresent teachings can be more fully understood from the followingdescription in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The skilled artisan will understand that the figures, described herein,are for illustration purposes only. It is to be understood that in someinstances various aspects of the invention may be shown exaggerated orenlarged to facilitate an understanding of the invention. In thedrawings, like reference characters generally refer to like features,functionally similar and/or structurally similar elements throughout thevarious figures. The drawings are not necessarily to scale, emphasisinstead being placed upon illustrating the principles of the teachings.The drawings are not intended to limit the scope of the presentteachings in any way.

The features and advantages of the present invention will become moreapparent from the detailed description set forth below when taken inconjunction with the drawings.

When describing embodiments in reference to the drawings, directionreferences (“above,” “below,” “top,” “bottom,” “left,” “right,”“horizontal,” “vertical,” etc.) may be used.

Such references are intended merely as an aid to the reader viewing thedrawings in a normal orientation. These directional references are notintended to describe a preferred or only orientation of an embodieddevice. A device may be embodied in other orientations.

FIG. 1-1 depicts a plot of probability for emitting a photon from amarker as a function of time.

FIG. 1-2A depicts emission timing spectra, according to someembodiments.

FIG. 1-2B depicts absorption wavelength spectra, according to someembodiments.

FIG. 1-2C depicts emission wavelength spectra, according to someembodiments.

FIG. 1-3A depicts a phase space for emission wavelength and emissionlifetime.

FIG. 1-3B depicts a phase space for absorption wavelength and emissionlifetime.

FIG. 1-4 depicts a phase space for emission wavelength, absorptionwavelength, and emission lifetime.

FIG. 2-1A is a block diagram representation of an apparatus that may beused for rapid, mobile analysis of biological and chemical specimens,according to some embodiments.

FIG. 2-1B is a block diagram of an integrated device and an instrument,according to some embodiments.

FIG. 2-2 depicts an integrated device, according to some embodiments.

FIG. 3-1A depicts a row of pixels of an integrated device, according tosome embodiments.

FIG. 3-1B depicts excitation energy coupling to sample wells in a row ofpixels and emission energy from each sample well directed towardssensors, according to some embodiments.

FIG. 3-2 depicts an integrated device and an excitation source,according to some embodiments.

FIG. 4-1A depicts edge-coupling of an excitation source to a waveguide,according to some embodiments.

FIG. 4-1B depicts a grating coupler for coupling an integrated device toan excitation source, according to some embodiments.

FIG. 4-2 depicts an integrated device and an excitation source,according to some embodiments.

FIG. 4-3A depicts an exemplary excitation coupling region, according tosome embodiments.

FIG. 4-3B depicts simulations of light intensity for the excitationcoupling region shown in FIG. 4-3A.

FIG. 4-3C depicts a grating coupler and waveguide, according to someembodiments.

FIG. 4-4 plots loss as a function of bend radius for different waveguideconfigurations.

FIG. 4-5 depicts a star coupler, according to some embodiments.

FIG. 4-6 depicts a star coupler for coupling input light from twograting couplers, according to some embodiments.

FIG. 4-7 depicts configurations for MMI splitters, according to someembodiments.

FIG. 4-8 depicts simulations of light intensity through a MMI splitter.

FIG. 4-9A depicts a grating coupler, according to some embodiments.

FIGS. 4-9B and 4-9C depicts a grating coupler, according to someembodiments.

FIG. 5-1 depicts a sample well formed in a pixel region of an integrateddevice, according to one embodiment.

FIG. 5-2 depicts excitation energy incident on a sample well, accordingto some embodiments.

FIG. 5-3 illustrates attenuation of excitation energy along a samplewell that is formed as a zero-mode waveguide, according to someembodiments.

FIG. 5-4 depicts a sample well that includes a divot, which increasesexcitation energy at an excitation region associated with the samplewell in some embodiments.

FIG. 5-5 compares excitation intensities for sample wells with andwithout a divot, according to one embodiment.

FIG. 5-6A depicts a sample well and divot formed at a protrusion,according to some embodiments.

FIG. 5-6B depicts a sample well and divot, according to someembodiments.

FIG. 5-7A depicts a sample well having tapered sidewalls, according tosome embodiments.

FIG. 5-7B depicts a sample well having curved sidewalls and a divot witha smaller transverse dimension, according to some embodiments.

FIG. 5-7C and FIG. 5-7D depict a sample well formed from surfaceplasmonic structures.

FIG. 5-7E depicts a sample well that includes anexcitation-energy-enhancing structure formed along sidewalls of thesample well, according to some embodiments.

FIG. 5-7F depicts a sample well formed in a multi-layer stack, accordingto some embodiments.

FIG. 5-8 illustrates surface coating formed on surfaces of a samplewell, according to some embodiments.

FIG. 5-9A through FIG. 5-9E depict structures associated with a lift-offprocess of forming a sample well, according to some embodiments.

FIG. 5-9F depicts a structure associated with an alternative lift-offprocess of forming a sample well, according to some embodiments.

FIG. 5-10A through FIG. 5-10D depict structures associated with a directetching process of forming a sample well, according to some embodiments.

FIG. 5-11 depicts a sample well that may be formed in multiple layersusing a lift-off process or a direct etching process, according to someembodiments.

FIG. 5-12 depicts a structure associated with an etching process thatmay be used to form a divot, according to some embodiments.

FIG. 5-13A through FIG. 5-13C depict structures associated with analternative process of forming a divot, according to some embodiments.

FIG. 5-14A through FIG. 5-14D depict structures associated with aprocess for depositing an adherent and passivating layers, according tosome embodiments.

FIG. 5-15 depicts a structure associated with a process for depositingan adherent centrally within a sample well, according to someembodiments.

FIG. 5-16 depicts a sample well with a divot, according to someembodiments.

FIG. 6-1A depicts a simulation of excitation radiation from a waveguidecoupled to a sample well, according to some embodiments.

FIG. 6-1B depicts a simulation of excitation radiation coupled to asample well, according to some embodiments.

FIGS. 6-2A, 6-2B, and 6-2C depict an integrated device withmicrocavities, according to some embodiments.

FIG. 6-3A depicts an integrated device with a microcavity, according tosome embodiments.

FIG. 6-3B depicts an integrated device with microcavities, according tosome embodiments.

FIG. 6-3C depicts an integrated device with a microcavity, according tosome embodiments.

FIG. 6-3D depicts an integrated device with microcavities, according tosome embodiments.

FIG. 6-4 depicts an integrated device with a microcavity, according tosome embodiments.

FIG. 6-5 depicts a simulation of excitation radiation propagating in anintegrated device with a microcavity, according to some embodiments.

FIGS. 6-6A, 6-6B, and 6-6C depict a simulation of excitation radiationpropagating in an integrated device with a microcavity, according tosome embodiments.

FIG. 6-6D depict a simulation of excitation radiation propagating in anintegrated device with a microcavity, according to some embodiments.

FIG. 6-7A depicts an integrated device with a microcavity, according tosome embodiments.

FIG. 6-7B depicts a simulation of excitation radiation propagating in anintegrated device with a microcavity, according to some embodiments.

FIG. 6-7C depicts a simulation of excitation radiation propagating in anintegrated device with a microcavity, according to some embodiments.

FIG. 6-7D depicts an integrated device with a sample well, a waveguide,and a microcavity, according to some embodiments.

FIGS. 6-8A and 6-8B depict an integrated device with a taperedwaveguide, according to some embodiments.

FIGS. 6-9A and 6-9B depict an integrated device with a taperedwaveguide, according to some embodiments.

FIG. 6-10 depicts a plot of loss as a function of taper length.

FIG. 6-11A depicts an integrated device with sample well dips, accordingto some embodiments.

FIGS. 6-11B and 6-11C depict an integrated device with sample well dips,according to some embodiments.

FIG. 6-12 depicts an array of sample wells of an integrated device,according to some embodiments.

FIG. 6-13 depicts an integrated device with a waveguide having avariable dimension, according to some embodiments.

FIG. 6-14 depicts an integrated device with a waveguide having avariable dimension, according to some embodiments.

FIGS. 7-1A, 7-1B, and 7-1C depict components to couple emission energyfrom a sample well of an integrated device, according to someembodiments.

FIG. 7-2A depicts a simulation of emission energy from a sample well.

FIG. 7-2B depicts a plot of emission energy at an angle from a samplewell.

FIG. 7-3 depicts a plot of absorptance and reflectance as a function ofwavelength.

FIGS. 7-4A and 7-4B depict a polarization filter, according to someembodiments.

FIG. 7-5 depicts a wavelength filter, according to some embodiments.

FIG. 7-6 depicts a plot of transmittance as a function of wavelength.

FIG. 7-7 depicts a multi-wavelength filter, according to someembodiments.

FIG. 7-8 depicts a plot of transmittance as a function of wavelength.

FIGS. 7-9A and 7-9B depict a sensor with time bins, according to someembodiments.

FIG. 8-0A depicts an exemplary system for providing light pulses,according to some embodiments.

FIG. 8-0B depicts a plot of light intensity as a function of time.

FIG. 8-1 depicts a plot of carrier density as a function of time.

FIG. 8-2 depicts a tailored electrical signal to form an optical output,according to some embodiments.

FIG. 8-3 depicts an optical output from an excitation source, accordingto some embodiments.

FIG. 8-4 depicts an optical output from an excitation source, accordingto some embodiments.

FIG. 8-5 depicts performance of a laser diode, according to someembodiments.

FIG. 8-6A depicts a transmission line pulsar, according to someembodiments.

FIG. 8-6B depicts light pulses obtained by a transmission line pulsar,according to some embodiments.

FIG. 8-7 depicts a circuit for obtaining light pulses, according to someembodiments.

FIG. 8-8 depicts a circuit for obtaining light pulses, according to someembodiments.

FIG. 8-9A depicts a circuit for obtaining light pulses, according tosome embodiments.

FIG. 8-9B depicts an electrical signal from the circuit shown in FIG.8-9A.

FIG. 8-10A depicts a circuit for obtaining light pulses, according tosome embodiments.

FIG. 8-10B depicts an electrical signal from the circuit shown in FIG.8-10A.

FIG. 8-11A depicts an arrangement for combining light sources, accordingto some embodiments.

FIG. 8-11B depicts a plot of performance of the circuit shown in FIG.8-10A.

FIG. 9-1 depicts an excitation source module and base instrument,according to some embodiments.

FIG. 9-2 depicts an excitation source module and base instrument,according to some embodiments.

FIG. 9-3 depicts optical components for aligning an excitation source toan integrated device, according to some embodiments.

FIG. 9-4 depicts an excitation source module and base instrument,according to some embodiments.

FIG. 9-5 through 9-11 depicts an excitation source module and baseinstrument, according to some embodiments.

FIG. 9-12 through 9-19 depicts components for passive alignment of anexcitation source to an integrated device, according to someembodiments.

FIG. 9-20 depicts monitoring sensors, according to some embodiments.

FIG. 9-21 depicts an integrated device with monitoring sensors,according to some embodiments.

FIG. 9-22 depicts an arrangement of waveguides and monitoring sensors ofan integrated device, according to some embodiments.

FIG. 9-23 depicts monitoring sensors of an integrated device, accordingto some embodiments.

FIG. 9-24 depicts optical components for coupling the excitation energyto the integrated device, according to some embodiments.

FIG. 9-25A depicts components for coupling the excitation energy to theintegrated device, according to some embodiments.

FIG. 9-25B depicts components for coupling the excitation energy to theintegrated device, according to some embodiments.

FIG. 9-25C depicts components for coupling the excitation energy to theintegrated device, according to some embodiments.

FIG. 10-1 shows a schematic of a sample well containing variouscomponents for nucleic acid sequencing, showing a target volume,polymerase complex, target nucleic acid, complimentary strand andprimer, and linker for immobilizing.

FIG. 10-2 shows an exemplary experiment of nucleic acid sequencing forfour stages of a sequencing reaction; (A) before incorporation of aluminescently labeled nucleotide; (B) a first incorporation event; (C) aperiod between the first and second incorporation events; and (D) asecond incorporation event; along with corresponding examples of raw andprocessed data during stages (A)-(D).

FIG. 10-3 shows an exemplary process for surface preparation, includingthe steps of (a) Al2O3 deposition, (b) PEG-phosphonate passivation, (c)Biotin/PEG-silanization, (d) complex loading, and (e) sequencingreaction initiation.

FIG. 10-4 depicts a schematic for performing measurements, according tosome embodiments.

FIG. 10-5 depicts a Fresnel lens, according to some embodiments.

FIG. 10-6 depicts a plot of light signal as a function of time,according to some embodiments.

FIG. 10-7 depicts a signal profile for markers across time bins,according to some embodiments.

FIG. 10-8 depicts a plot of light signal as a function of time,according to some embodiments.

FIG. 10-9 depicts a signal profile for markers across time bins,according to some embodiments.

FIG. 10-10 depicts a schematic for performing measurements, according tosome embodiments.

FIG. 10-11 depicts a plot of lifetime as a function of emissionwavelength, according to some embodiments.

FIG. 10-12 depicts a plot of light signal as a function of wavelength,according to some embodiments.

FIG. 10-13 depicts a plot of light signal as a function of time,according to some embodiments.

FIG. 10-14 depicts a signal profile for markers across time bins formultiple sensors, according to some embodiments.

FIG. 10-15 depicts a plot of light signal as a function of time,according to some embodiments.

FIG. 10-16 depicts a signal profile for a marker across time bins formultiple sensors, according to some embodiments.

FIG. 10-17 depicts a schematic for performing measurements, according tosome embodiments.

FIG. 10-18 depicts a plot of light signal as a function of wavelength,according to some embodiments.

FIG. 10-19 depicts a plot of light signal as a function of time,according to some embodiments.

FIG. 10-20 depicts a signal profile for a marker across time bins formultiple sensors, according to some embodiments.

FIG. 11-1 depicts a method for fabricating a sample well, according tosome embodiments.

FIG. 11-2 depicts a method for fabricating a sample well, according tosome embodiments.

FIG. 11-3 depicts a method for fabricating a sample well, according tosome embodiments.

FIG. 11-4A depicts a method for fabricating a sample well, according tosome embodiments.

FIG. 11-4B depicts a method for fabricating a sample well, according tosome embodiments.

FIG. 11-5 depicts a method for fabricating a sample well layer withdips, according to some embodiments.

FIG. 11-6 depicts a method for fabricating a sample well layer withdips, according to some embodiments.

FIG. 11-7 depicts a method for fabricating a concentric grating,according to some embodiments.

FIG. 11-8 depicts a method for fabricating a concentric grating,according to some embodiments.

FIG. 11-9 depicts a method for fabricating a concentric grating,according to some embodiments.

FIG. 11-10 depicts exemplary microcavity designs, according to someembodiments.

FIG. 11-11 depicts a method for fabricating refractive optics, accordingto some embodiments.

FIG. 11-12 depicts images of different steps in fabricating refractiveoptics, according to some embodiments.

FIG. 11-13 depicts a refractive optic, according to some embodiments.

FIG. 11-14 depicts a method for fabricating refractive optics, accordingto some embodiments.

FIG. 11-15 depicts a method for fabricating refractive optics, accordingto some embodiments.

FIG. 11-16 depicts a Fresnel lens, according to some embodiments.

FIG. 11-17 depicts a Fresnel lens, according to some embodiments.

FIG. 11-18 depicts a Fresnel lens, according to some embodiments.

FIG. 11-19 depicts a method for fabricating a Fresnel lens, according tosome embodiments.

FIG. 11-20 depicts a method for fabricating a Fresnel lens, according tosome embodiments.

FIG. 11-21 depicts a method for fabricating a Fresnel lens, according tosome embodiments.

FIG. 11-22 depicts a method for fabricating a Fresnel lens, according tosome embodiments.

DETAILED DESCRIPTION

The inventors have recognized and appreciated that a compact, high-speedapparatus for performing detection and quantitation of single moleculesor particles could reduce the cost of performing complex quantitativemeasurements of biological and/or chemical samples and rapidly advancethe rate of biochemical technological discoveries. Moreover, acost-effective device that is readily transportable could transform notonly the way bioassays are performed in the developed world, but providepeople in developing regions, for the first time, access to essentialdiagnostic tests that could dramatically improve their health andwell-being. For example, embodiments described herein may be used fordiagnostic tests of blood, urine and/or saliva that may be used byindividuals in their home, or by a doctor in a remote clinic in adeveloping country.

A pixelated sensor device with a large number of pixels (e.g., hundreds,thousands, millions or more) allows for the detection of a plurality ofindividual molecules or particles in parallel. The molecules may be, byway of example and not limitation, proteins and/or DNA. Moreover, ahigh-speed device that can acquire data at more than one hundred framesper second allows for the detection and analysis of dynamic processes orchanges that occur over time within the sample being analyzed.

The inventors have recognized and appreciated that one hurdle preventingbioassay equipment from being made more compact was the need to filterthe excitation light from causing undesirable detection events at thesensor. Optical filters used to transmit the desired signal light (theluminescence) and sufficiently block the excitation light can be thick,bulky, expensive, and intolerant to variations in the incidence angle oflight, preventing miniaturization. The inventors, however, recognizedand appreciated that using a pulsed excitation source can reduce theneed for such filtering or, in some cases, remove the need for suchfilters altogether. By using sensors capable of determining the time aphoton is detected relative to the excitation light pulse, the signallight can be separated from the excitation light based on the time thatthe photon is received, rather than the spectrum of the light received.Accordingly, the need for a bulky optical filter is reduced and/orremoved in some embodiments.

The inventors have recognized and appreciated that luminescence lifetimemeasurements may also be used to identify the molecules present in asample. An optical sensor capable of detecting when a photon is detectedis capable of measuring, using the statistics gathered from many events,the luminescence lifetime of the molecule being excited by theexcitation light. In some embodiments, the luminescence lifetimemeasurement may be made in addition to a spectral measurement of theluminescence. Alternatively, a spectral measurement of the luminescencemay be completely omitted in identifying the sample molecule.Luminescence lifetime measurements may be made with a pulsed excitationsource. Additionally, luminescence lifetime measurements may be madeusing an integrated device that includes the sensor, or a device wherethe light source is located in a system separate from the integrateddevice.

The inventors have also recognized and appreciated that integrating asample well (which may include a nanoaperture) and a sensor in a singleintegrated device capable of measuring luminescent light emitted frombiological samples reduces the cost of producing such a device such thatdisposable bioanalytical integrated devices may be formed. Disposable,single-use integrated devices that interface with a base instrument maybe used anywhere in the world, without the constraint of requiringhigh-cost biological laboratories for sample analyses. Thus, automatedbioanalytics may be brought to regions of the world that previouslycould not perform quantitative analysis of biological samples. Forexample, blood tests for infants may be performed by placing a bloodsample on a disposable integrated device, placing the disposableintegrated device into a small, portable base instrument for analysis,and processing the results by a computer for immediate review by a user.The data may also be transmitted over a data network to a remotelocation to be analyzed, and/or archived for subsequent clinicalanalyses.

The inventors have also recognized and appreciated that a disposable,single-use device may be made more simply and for lower cost by notincluding the light source on the integrated device. Instead, the lightsource may include reusable components incorporated into a system thatinterfaces with the disposable integrated device to analyze a sample.

The inventors have also recognized and appreciated that, when a sampleis tagged with a plurality of different types of luminescent markers,any suitable characteristic of luminescent markers may be used toidentify the type of marker that is present in a particular pixel of theintegrated device. For example, characteristics of the luminescenceemitted by the markers and/or characteristics of the excitationabsorption may be used to identify the markers. In some embodiments, theemission energy of the luminescence (which is directly related to thewavelength of the light) may be used to distinguish a first type ofmarker from a second type of marker. Additionally, or alternatively,luminescence lifetime measurements may also be used to identify the typeof marker present at a particular pixel. In some embodiments,luminescence lifetime measurements may be made with a pulsed excitationsource using a sensor capable of distinguishing a time when a photon isdetected with sufficient resolution to obtain lifetime information.Additionally, or alternatively, the energy of the excitation lightabsorbed by the different types of markers may be used to identify thetype of marker present at a particular pixel. For example, a firstmarker may absorb light of a first wavelength, but not equally absorblight of a second wavelength, while a second marker may absorb light ofthe second wavelength, but not equally absorb light of the firstwavelength. In this way, when more than one excitation light source,each with a different excitation energy, may be used to illuminate thesample in an interleaved manner, the absorption energy of the markerscan be used to identify which type of marker is present in a sample.Different markers may also have different luminescent intensities.Accordingly, the detected intensity of the luminescence may also be usedto identify the type of marker present at a particular pixel.

One non-limiting example of an application of a device contemplated bythe inventors is a device capable of performing sequencing of abiomolecule, such as a nucleic acid or a polypeptide (e.g. protein)having a plurality of amino acids. Diagnostic tests that may beperformed using such a device include sequencing a nucleic acid moleculein a biological sample of a subject, such as sequencing of cell freedeoxyribonucleic acid molecules or expression products in a biologicalsample of the subject.

The present application provides devices, systems and methods fordetecting biomolecules or subunits thereof, such as nucleic acidmolecules. Such detection can include sequencing. A biomolecule may beextracted from a biological sample obtained from a subject. Thebiological sample may be extracted from a bodily fluid or tissue of thesubject, such as breath, saliva, urine or blood (e.g., whole blood orplasma). The subject may be suspected of having a health condition, suchas a disease (e.g., cancer). In some examples, one or more nucleic acidmolecules are extracted from the bodily fluid or tissue of the subject.The one or more nucleic acids may be extracted from one or more cellsobtained from the subject, such as part of a tissue of the subject, orobtained from a cell-free bodily fluid of the subject, such as wholeblood.

Sequencing can include the determination of individual subunits of atemplate biomolecule (e.g., nucleic acid molecule) by synthesizinganother biomolecule that is complementary or analogous to the template,such as by synthesizing a nucleic acid molecule that is complementary toa template nucleic acid molecule and identifying the incorporation ofnucleotides with time (e.g., sequencing by synthesis). As analternative, sequencing can include the direct identification ofindividual subunits of the biomolecule.

During sequencing, signals indicative of individual subunits of abiomolecule may be collected in memory and processed in real time or ata later point in time to determine a sequence of the biomolecule. Suchprocessing can include a comparison of the signals to reference signalsthat enable the identification of the individual subunits, which in somecases yields reads. Reads may be sequences of sufficient length (e.g.,at least about 30, 50, 100 base pairs (bp) or more) that can be used toidentify a larger sequence or region, e.g., that can be aligned to alocation on a chromosome or genomic region or gene.

Individual subunits of biomolecules may be identified using markers. Insome examples, luminescent markers are used to identify individualsubunits of biomolecules. Luminescent markers (also referred to hereinas “markers”) may be exogenous or endogenous markers. Exogenous markersmay be external luminescent markers used in a reporter and/or tag forluminescent labeling. Examples of exogenous markers may include, but arenot limited to, fluorescent molecules, fluorophores, fluorescent dyes,fluorescent stains, organic dyes, fluorescent proteins, enzymes, speciesthat participate in fluorescence resonance energy transfer (FRET),enzymes, and/or quantum dots. Such exogenous markers may be conjugatedto a probe or functional group (e.g., molecule, ion, and/or ligand) thatspecifically binds to a particular target or component. Attaching anexogenous marker to a probe allows identification of the target throughdetection of the presence of the exogenous marker. Examples of probesmay include proteins, nucleic acid (e.g. DNA, RNA) molecules, lipids andantibody probes. The combination of an exogenous marker and a functionalgroup may form any suitable probes, tags, and/or labels used fordetection, including molecular probes, labeled probes, hybridizationprobes, antibody probes, protein probes (e.g., biotin-binding probes),enzyme labels, fluorescent probes, fluorescent tags, and/or enzymereporters.

Although the present disclosure makes reference to luminescent markers,other types of markers may be used with devices, systems and methodsprovided herein. Such markers may include mass tags or electrostatictags.

While exogenous markers may be added to a sample, endogenous markers maybe already part of the sample. Endogenous markers may include anyluminescent marker present that may luminesce or “autofluoresce” in thepresence of excitation energy. Autofluorescence of endogenousfluorophores may provide for label-free and noninvasive labeling withoutrequiring the introduction of exogenous fluorophores. Examples of suchendogenous fluorophores may include hemoglobin, oxyhemoglobin, lipids,collagen and elastin crosslinks, reduced nicotinamide adeninedinucleotide (NADH), oxidized flavins (FAD and FMN), lipofuscin,keratin, and/or prophyrins, by way of example and not limitation.

While some embodiments may be directed to diagnostic testing bydetecting single molecules in a specimen, the inventors have alsorecognized that some embodiments may use the single molecule detectioncapabilities to perform nucleic acid (e.g. DNA, RNA) sequencing of oneor more nucleic acid segments such as, for example, genes, orpolypeptides. Nucleic acid sequencing allows for the determination ofthe order and position of nucleotides in a target nucleic acid molecule.Nucleic acid sequencing technologies may vary in the methods used todetermine the nucleic acid sequence as well as in the rate, read length,and incidence of errors in the sequencing process. For example, somenucleic acid sequencing methods are based on sequencing by synthesis, inwhich the identity of a nucleotide is determined as the nucleotide isincorporated into a newly synthesized strand of nucleic acid that iscomplementary to the target nucleic acid molecule. Some sequencing bysynthesis methods require the presence of a population of target nucleicacid molecules (e.g., copies of a target nucleic acid) or a step ofamplification of the target nucleic acid to achieve a population oftarget nucleic acids.

Having recognized the need for simple, less complex apparatuses forperforming single molecule detection and/or nucleic acid sequencing, theinventors have conceived of a technique for detecting single moleculesusing sets of markers, such as optical (e.g., luminescent) markers, tolabel different molecules. A tag may include a nucleotide or amino acidand a suitable marker. Markers may be detected while bound to singlemolecules, upon release from the single molecules, or while bound to andupon release from the single molecules. In some examples, markers areluminescent tags. Each luminescent marker in a selected set isassociated with a respective molecule. For example, a set of fourmarkers may be used to “label” the nucleobases present in DNA—eachmarker of the set being associated with a different nucleobase to form atag, e.g., a first marker being associated with adenine (A), a secondmarker being associated with cytosine (C), a third marker beingassociated with guanine (G), and a fourth marker being associated withthymine (T). Moreover, each of the luminescent markers in the set ofmarkers has different properties that may be used to distinguish a firstmarker of the set from the other markers in the set. In this way, eachmarker is uniquely identifiable using one or more of thesedistinguishing characteristics. By way of example and not limitation,the characteristics of the markers that may be used to distinguish onemarker from another may include the emission energy and/or wavelength ofthe light that is emitted by the marker in response to excitation and/orthe wavelength and/or energy of the excitation light that excites aparticular marker. Distinguishing a marker from among the set of fourmarkers uniquely identifies the nucleobase associated with the marker.

Luminescent markers may vary in the wavelength of light they emit, thetemporal characteristics of the light they emit (e.g., their emissiondecay time periods), and their response to excitation energy (e.g.,their probability of absorbing an excitation photon). Accordingly,luminescent markers may be identified or discriminated from otherluminescent markers based on detecting these properties. Suchidentification or discrimination techniques may be used alone or in anysuitable combination.

In some embodiments, an integrated photodetector as described in thepresent application can measure or discriminate luminescence lifetimes,such as fluorescence lifetimes. Lifetime measurements are based onexciting one or more markers (e.g., fluorescent molecules), andmeasuring the time variation in the emitted luminescence. Theprobability that a marker emits a photon after the marker reaches anexcited state decreases exponentially over time. The rate at which theprobability decreases may be characteristic of a marker, and may bedifferent for different markers. Detecting the temporal characteristicsof light emitted by markers may allow identifying markers and/ordiscriminating markers with respect to one another. The decrease in theprobability of a photon being emitted over time may be represented by anexponential decay function p(t)=e{circumflex over ( )}(−t/τ), where p(t)is the probability of photon emission at a time, t, and τ is a temporalparameter of the marker. The temporal parameter τ indicates a time afterexcitation when the probability of the marker emitting a photon is acertain value. The temporal parameter, τ, is a property of a marker thatmay be distinct from its absorption and emission spectral properties.Such a temporal parameter, τ, is referred to as the luminescencelifetime, the fluorescence lifetime or simply the “lifetime” of amarker.

FIG. 1-1 plots the probability of a photon being emitted as a functionof time for two markers with different lifetimes. The marker representedby probability curve B has a probability of emission that decays morequickly than the probability of emission for the marker represented byprobability curve A. The marker represented by probability curve B has ashorter temporal parameter, τ, or lifetime than the marker representedby probability curve A. Markers may have lifetimes ranging from 0.1-20ns, in some embodiments. However, the techniques described herein arenot limited as to the lifetimes of the marker(s) used.

The lifetime of a marker may be used to distinguish among more than onemarker, and/or may be used to identify marker(s). In some embodiments,lifetime measurements may be performed in which a plurality of markershaving different lifetimes is excited by an excitation source. As anexample, four markers having lifetimes of 0.5, 1, 2, and 3 nanoseconds,respectively, may be excited by a light source that emits light having aselected wavelength (e.g., 635 nm, by way of example). The markers maybe identified or differentiated from each other based on measuring thelifetime of the light emitted by the markers.

Lifetime measurements may use relative intensity measurements bycomparing how intensity changes over time, as opposed to absoluteintensity values. As a result, lifetime measurements may avoid some ofthe difficulties of absolute intensity measurements. Absolute intensitymeasurements may depend on the concentration of markers present andcalibration steps may be needed for varying marker concentrations. Bycontrast, lifetime measurements may be insensitive to the concentrationof markers.

Embodiments may use any suitable combination of marker characteristicsto distinguish a first marker in a set of markers from the other markersin the same set. For example, some embodiments may use only the timinginformation of the emission light from the markers to identify themarkers. In such embodiments, each marker in a selected set of markershas a different emission lifetime from the other markers in the set andthe luminescent markers are all excited by light from a singleexcitation source. FIG. 1-2A illustrates the emission timing from fourluminescent markers according to an embodiment where the four markersexhibit different average emission lifetimes (t). The probability that amarker is measured to have a lifetime of a particular value is referredto herein as the marker's “emission timing.” A first emission timing1-101 from a first luminescent marker has a peak probability of having alifetime of at τ1, a second emission timing 1-102 from a secondluminescent marker has a peak probability of having a lifetime of at τ2,a third emission timing 1-103 from a third luminescent marker has a peakprobability of having a lifetime of at τ3, and a fourth emission timing1-104 from a fourth luminescent marker has a peak probability of havinga lifetime of at τ4. In this embodiment, the lifetime probability peaksof the four luminescent markers may have any suitable values thatsatisfy the relation τ1<τ2<τ3<τ4. The four timing emission graphs may ormay not overlap due to slight variations in the lifetime of a particularluminescent marker, as illustrated in FIG. 1-2A. In this embodiment, theexcitation wavelength at which each of the four markers maximallyabsorbs light from the excitation source is substantially equal, butthat need not be the case. Using the above marker set, four differentmolecules may be labeled with a respective marker from the marker set,the markers may be excited using a single excitation source, and themarkers can be distinguished from one another by detecting the emissionlifetime of the markers using an optical system and sensors. While FIG.1-2A illustrates four different markers, it should be appreciated thatany suitable number of markers may be used.

Other embodiments may use any suitable combination of markercharacteristics to determine the identity of the marker within a set ofmarkers. Examples of the marker characteristics that may be usedinclude, but are not limited to excitation wavelength, emissionwavelength, and emission lifetime. The combination of markercharacteristics form a phase space and each marker may be represented asa point within this phase space. Markers within a set of markers shouldbe selected such that the “distance” between each marker within the setis sufficiently large that the detection mechanism can distinguish eachmarker from the other markers in the set. For example, in someembodiments a set of markers may be selected where a subset of themarkers have the same emission wavelength, but have different emissionlifetimes and/or different excitation wavelengths. In other embodiments,a set of markers may be selected where a subset of the markers have thesame emission lifetime, but have different emission wavelengths and/ordifferent excitation wavelengths. In other embodiments, a set of markersmay be selected where a subset of the markers have the same excitationwavelength, but have different emission wavelengths and/or differentemission lifetimes.

By way of example and not limitation, FIG. 1-2B illustrates the emissionspectra from four luminescent markers according to an embodiment wheretwo of the markers have a first peak emission wavelength and the othertwo markers have a second peak emission wavelength. A first emissionspectrum 1-105 from a first luminescent marker has a peak emissionwavelength at λ1, a second emission spectrum 1-106 from a secondluminescent marker also has a peak emission wavelength at λ1, a thirdemission spectrum 1-107 from a third luminescent marker has a peakemission wavelength at λ2, and a fourth emission spectrum 1-108 from afourth luminescent marker also has a peak emission wavelength at λ2. Inthis embodiment, the emission peaks of the four luminescent markers mayhave any suitable values that satisfy the relation λ1<λ2. In embodimentssuch as this where the peak emission wavelength is the same for morethan one luminescent marker, a separate characteristic of the markersthat have the same emission wavelength must be different. For example,the two markers that emit at λ1 may have different emission lifetimes.FIG. 1-3A illustrates this situation schematically in a phase spacespanned by the emission wavelength and the emission lifetime. A firstmarker has an emission wavelength λ1 and an emission lifetime Ti, asecond marker has an emission wavelength λ1 and a emission lifetime τ4,a third marker has an emission wavelength λ2 and a emission lifetime τ1,and a fourth marker has an emission wavelength λ2 and a emissionlifetime τ4. In this way, all four markers in the marker set shown inFIG. 1-3A are distinguishable from one another. Using such a marker setallows distinguishing between four markers even when the absorptionwavelengths for the four markers are identical. This is possible using asensor that can detect the time of emission of the photoluminescence aswell as the emission wavelength.

By way of example and not limitation, FIG. 1-2C illustrates theabsorption spectra from four luminescent markers according to anotherembodiment. In this embodiment, two of the markers have a first peakabsorption wavelength and the other two markers have a second peakabsorption wavelength. A first absorption spectrum 1-109 for the firstluminescent marker has a peak absorption wavelength at λ3, a secondabsorption spectrum 1-110 for the second luminescent marker has a peakabsorption wavelength at λ4, a third absorption spectrum 1-111 for thethird luminescent marker has a peak absorption wavelength at λ3, and afourth absorption spectrum 1-112 for the fourth luminescent marker has apeak absorption wavelength at λ4. Note that the markers that share anabsorption peak wavelength in FIG. 1-2C are distinguishable via anothermarker characteristic, such as emission lifetime. FIG. 1-3B illustratesthis situation schematically in a phase space spanned by the absorptionwavelength and the emission lifetime. A first marker has an absorptionwavelength λ3 and an emission lifetime τ1, a second marker has anabsorption wavelength λ3 and an emission lifetime τ4, a third marker hasan absorption wavelength λ4 and an emission lifetime τ1, and a fourthmarker has an absorption wavelength λ4 and an emission lifetime τ4. Inthis way, all four markers in the marker set shown in FIG. 1-3A aredistinguishable from one another.

Using such a marker set allows distinguishing between four markers evenwhen the emission wavelengths for the four markers areindistinguishable. This is possible using two excitation sources thatemit at different wavelengths or a single excitation source capable ofemitting at multiple wavelengths in connection with a sensor that candetect the time of emission of the photoluminescence. If the wavelengthof the excitation light is known for each detected emission event, thenit can be determined which marker was present. The excitation source(s)may alternate between a first excitation wavelength and a secondexcitation wavelength, which is referred to as interleaving.Alternatively, two or more pulses of the first excitation wavelength maybe used followed by two or more pulses of the second excitationwavelength.

The number of excitation sources or excitation wavelengths used todistinguish the markers is not limited to two, and in some embodimentsmore than two excitation wavelengths or energies may be used todistinguish the markers. In such embodiments, markers may bedistinguished by the intensity or number of photons emitted in responseto multiple excitation wavelengths. A marker may be distinguishable fromamong multiple markers by detecting the number of photons emitted inresponse to exposing the marker to a certain excitation wavelength. Insome embodiments, a marker may be distinguished by illuminating themarker to one of multiple excitation energies at a time and identifyingthe excitation energy from among the multiple excitation energies wherethe marker emitted the highest number of photons. In other embodiments,the number of photons emitted from a marker in response to differentexcitation energies may be used to identify the marker. A first markerthat has a higher probability of emitting photons in response to a firstexcitation energy than a second excitation energy may be distinguishedfrom a second marker that has a higher probability of emitting photonsin response to the second excitation energy than the first excitationenergy. In this manner, markers having distinguishable probabilities ofemitting certain amounts of photons in response to different excitationenergies may be identified by measuring the emitted photons whileexposing an unknown marker to the different excitation energies. In suchembodiments, a marker may be exposed to multiple excitation energies andidentification of the marker may be achieved by determining whether themarker emitted any light and/or a particular number of photons emitted.Any suitable number of excitation energy sources may be used. In someembodiments, four different excitation energies may be used todistinguish among different markers (e.g., four different markers). Insome embodiments, three different excitation energies may be used todistinguish among different markers. Other characteristics of a markermay be used to distinguish the presence of a marker in combination withthe amount of photons emitted in response to different excitationenergies, including emission lifetime and emission spectra.

In other embodiments more than two characteristics of the markers in amarker set may be used to distinguish which marker is present. FIG. 1-4illustrates an illustrative phase space spanned by the absorptionwavelength, the emission wavelength and the emission lifetime of themarkers. In FIG. 1-4, eight different markers are distributed in phasespace. Four of the eight markers have the same emission wavelength, adifferent four markers have the same absorption wavelength and adifferent four markers have the same emission lifetime. However, each ofthe markers is distinguishable from every other marker when all threecharacteristics of the markers are considered. Embodiments are notlimited to any number of markers. This concept can be extended toinclude any number of markers that may be distinguished from one anotherusing at least these three marker characteristics.

While not illustrated in the figures, other embodiments may determinethe identity of a luminescent marker based on the absorption frequencyalone. Such embodiments are possible if the excitation light can betuned to specific wavelengths that match the absorption spectrum of themarkers in a marker set. In such embodiments, the optical system andsensor used to direct and detect the light emitted from each marker doesnot need to be capable of detecting the wavelength of the emitted light.This may be advantageous in some embodiments because it reduces thecomplexity of the optical system and sensors because detecting theemission wavelength is not required in such embodiments.

As discussed above, the inventors have recognized and appreciated theneed for being able to distinguish different luminescent markers fromone another using various characteristics of the markers. The type ofcharacteristics used to determine the identity of a marker impacts thephysical device used to perform this analysis. The present applicationdiscloses several embodiments of an apparatus, device, instrument andmethods for performing these different experiments.

The inventors have recognized and appreciated that a low-cost,single-use disposable integrated device that includes optics and sensorsmay be used in connection with an instrument that includes an excitationsource to measure different characteristics of luminescent light emittedfrom one or markers used to label a biological sample in order toanalyze the sample. Using a low-cost integrated device reduces the costof performing a given bioassay. A biological sample is placed onto theintegrated device and, upon completion of the bioassay, may bediscarded. The integrated device interfaces with the more expensive,multi-use instrument, which may be used repeatedly with many differentdisposable integrated devices. A low-cost integrated device thatinterfaces with a compact, portable instrument may be used anywhere inthe world, without the constraint of high-cost biological laboratoriesrequiring laboratory expertise to analyze samples. Thus, automatedbioanalytics may be brought to regions of the world that previouslycould not perform quantitative analysis of biological samples. Forexample, blood tests for infants may be performed by placing a bloodsample on a disposable integrated device, placing the disposableintegrated device into the small, portable instrument for analysis, andprocessing the results by a computer that connects to the instrument forimmediate review by a user. The data may also be transmitted over a datanetwork to a remote location to be analyzed, and/or archived forsubsequent clinical analyses. Alternatively, the instrument may includeone or more processors for analyzing the data obtained from the sensorsof the integrated device.

Various embodiments are described in more detail below.

I. Overview of the System

The system includes an integrated device and an instrument configured tointerface with the integrated device. The integrated device includes anarray of pixels, where a pixel includes a sample well and at least onesensor. A surface of the integrated device has a plurality of samplewells, where a sample well is configured to receive a sample from aspecimen placed on the surface of the integrated device. A specimen maycontain multiple samples, and in some embodiments, different types ofsamples. The plurality of sample wells may be designed such that atleast a portion of the sample wells are configured to receive one samplefrom a specimen. In some embodiments, the number of samples within asample well may be distributed among the sample wells such that somesample wells contain one sample with others contain zero, two or moresamples. For example, a specimen may contain multiple single-strandedDNA templates, and a sample well on a surface of an integrated devicemay receive a single-stranded DNA template. At least a portion of thesample wells of the integrated device may contain a single-stranded DNAtemplate. The specimen may also contain tagged dNTPs which then enter inthe sample well and may allow for identification of a nucleotide as itis incorporated into a complementary strand of DNA. In such an example,the “sample” may refer to both the single-stranded DNA and the taggeddNTP currently being incorporated by a polymerase. In some embodiments,the specimen may contain single-stranded DNA templates and tagged dNTPSmay be subsequently introduced to a sample well as nucleotides areincorporated into a complementary strand of DNA within the sample well.In this manner, timing of incorporation of nucleotides may be controlledby when tagged dNTPs are introduced to the sample wells of an integrateddevice.

Excitation energy is provided from an excitation source located separatefrom the pixel array of the integrated device. The excitation energy isdirected at least in part by elements of the integrated device towardsone or more pixels to illuminate an illumination region within thesample well. A marker or tag may then emit emission energy when locatedwithin the illumination region and in response to being illuminated byexcitation energy. In some embodiments, one or more excitation sourcesare part of the instrument of the system where components of theinstrument and the integrated device are configured to direct theexcitation energy towards one or more pixels. In other embodiments, oneor more excitation sources are located on the integrated device but arelocated in a separate region from the array of pixels, and components inthe integrated device are configured to direct excitation energy fromthe excitation source region to one or more pixels.

Emission energy emitted by a sample may then be detected by one or moresensors within a pixel of the integrated device. In some embodiments, aplurality of sensors may be sized and arranged to capture a spatialdistribution of the emission energy. In some embodiments one or moresensors may be configured to detect a timing characteristics associatedwith a sample's emission energy (e.g., fluorescence lifetime). Outputsignals from the one or more sensors may then be used to distinguish amarker from among a plurality of markers, where the plurality of markersmay be used to identify a sample within the specimen. In someembodiments, a sample may be excited by multiple excitation energies,and emission energy and/or timing characteristics of the emission energyemitted by the sample in response to the multiple excitation energiesmay distinguish a marker from a plurality of markers.

A schematic overview of the system 2-100 is illustrated in FIGS. 2-1Aand 2-1B. The system comprises both an integrated device 2-102 thatinterfaces with an instrument 2-104. In some embodiments, instrument2-104 may include one or more excitation source 2-106. In someembodiments, an excitation source may be external to both instrument2-104 and integrated device 2-102, and instrument 2-104 may beconfigured to receive excitation energy from the excitation source anddirect it to the integrated device. The integrated device interfaceswith the instrument using any suitable socket for receiving theintegrated device and holding it in precise optical alignment with theexcitation source. The excitation source 2-106 may be configured toprovide excitation energy to the integrated device 2-102. Although theexcitation source is shown to be located on the instrument in FIG. 2-1B,the excitation source may be located on the integrated device in aregion separate from the pixels in some instances. As illustratedschematically in FIG. 2-1B, the integrated device 2-102 has multiplepixels, where each pixel 2-112 is capable of independent analysis of asample. Such pixels 2-112 may be referred to as “passive source pixels”since a pixel receives excitation energy from a source 2-106 separatefrom the pixel, where the source excites a plurality of pixels. Eachpixel 2-112 has a sample well 2-108 for retaining and analyzing a sampleand a sensor 2-110 for detecting emission energy emitted by the samplein response to illuminating the sample with excitation energy providedby the excitation source 2-106. In some embodiments, each sensor 2-110may include multiple sub-sensors, each sub-sensor configured to detect adifferent wavelength of emission energy from the sample.

Optical elements for guiding and coupling excitation energy to thesample well 2-108 are located both on integrated device 2-102 and theinstrument 2-104. Such source-to-well elements may include a gratingcoupler located on integrated device 2-102 to couple excitation energyto the integrated device, waveguides to deliver excitation energy toeach pixel 2-112, and lenses, plasmonic elements and dielectric coatingson the integrated device to direct excitation energy received frominstrument 2-104 to sample well 2-108. Additionally, optical elementslocated on the integrated device direct emission energy from the samplewell towards the sensor. Such well-to-sample elements may includecomponents that direct the emission energy into a radiation patternwhere the radiation pattern depends on the emission energy emitted by asample in a sample well. Sample well 2-108, a portion of the excitationsource-to-well optics, and the sample well-to-sensor optics are locatedon integrated device 2-102. Excitation source 2-106 and a portion of thesource-to-well components are located in instrument 2-104. In someembodiments, a single component may play a role in both couplingexcitation energy to sample well 2-108 and delivering emission energyfrom sample well 2-108 to sensor 2-110.

As illustrated in FIG. 2-1B, the integrated device comprises a pluralityof pixels, each pixel 2-112 associated with its own individual samplewell 2-108 and sensor 2-110. The plurality of pixels may be arranged inan array, and there may be any suitable number of pixels. For example,integrated device 2-102 may include between 100 and 1,000 pixelsaccording to some embodiments, between 1,000 and 10,000 pixels accordingto some embodiments, between 10,000 and 100,000 pixels according to someembodiments, between 100,000 and 1,000,000 pixels according to someembodiments, and yet between 1,000,000 and 10,000,000 pixels accordingto some embodiments. In some implementations, there may be fewer or morepixels on integrated device 2-102. Integrated device 2-102 andinstrument 2-104 may include multi-channel, high-speed communicationlinks for handling data associated with large pixel arrays (e.g., morethan 1000 pixels).

Excitation source 2-106 may be any suitable source that is arranged todeliver excitation energy to at least one sample well. In someembodiments, an array of one or more excitation sources are locatedadjacent the pixel array on the same integrated device. In otherembodiments, one or more excitation sources are on a second substratemounted in close proximity to the substrate on which the pixel array isformed.

Instrument 2-104 interfaces with integrated device 2-102 throughintegrated device interface 2-114. Integrated device interface 2-114 mayinclude components to position and/or align integrated device 2-102 toinstrument 2-104 to improve coupling of excitation energy fromexcitation source 2-106 to integrated device 2-102. In some embodiments,excitation source 2-106 includes multiple excitation sources that arecombined to deliver excitation energy to integrated device 2-102. Themultiple excitation sources may be configured to produce multipleexcitation energies or wavelengths. The integrated device interface2-114 may receive readout signals from the sensors in the pixels locatedon the integrated device. Additionally, the integrated device interface2-114 may be designed such that the integrated device attaches to theinstrument by securing the integrated device to the integrated deviceinterface 2-114.

The instrument 2-104 includes a user interface 2-116 for controlling theoperation of instrument 2-104. The user interface 2-116 is configured toallow a user to input information into the instrument, such as commandsand/or settings used to control the functioning of the instrument. Insome embodiments, the user interface 2-116 may include buttons,switches, dials, and a microphone for voice commands. Additionally, theuser interface 2-116 may allow a user to receive feedback on theperformance of the instrument and/or integrated device, such as properalignment and/or information obtained by readout signals from thesensors on the integrated device. In some embodiments, the userinterface 2-116 may provide feedback using a speaker to provide audiblefeedback, and indicator lights and/or display screen for providingvisual feedback. In some embodiments, the instrument 2-104 includes acomputer interface 2-118 used to connect with a computing device 2-120.Any suitable computer interface 2-118 and computing device 2-120 may beused. For example, the computer interface 2-118 may be a USB interfaceor a FireWire interface. The computing device 2-120 may be any generalpurpose computer, such as a laptop or desktop computer. The computerinterface 2-118 facilitates communication of information between theinstrument 2-104 and the computing device 2-120. Input information forcontrolling and/or configuring the instrument 2-104 may be providedthrough the computing device 2-120 connected to the computer interface2-118 of the instrument. Additionally, output information may bereceived by the computing device 2-120 through the computer interface2-118. Such output information may include feedback about performance ofthe instrument 2-104 and/or or integrated device 2-112 and informationfrom the readout signals of the sensor 2-110. The instrument 2-104 mayalso include a processing device 2-122 for analyzing data received fromthe sensor 2-110 and/or sending control signals to the excitation source2-106. In some embodiments, the processing device 2-122 may comprise a ageneral purpose processor, a specially-adapted processor (e.g., acentral processing unit (CPU) such as one or more microprocessor ormicrocontroller cores, a field-programmable gate array (FPGA), anapplication-specific integrated circuit (ASIC), a custom integratedcircuit, a digital signal processor (DSP), or a combination thereof.) Insome embodiments, the processing of data from the sensor 2-110 may beperformed by both the processing device 2-122 and the external computingdevice 2-120. In other embodiments, the computing device 2-120 may beomitted and processing of data from the sensor 2-110 may be performedsolely by processing device 2-122.

A cross-sectional schematic of the integrated device 3-102 illustratinga row of pixels is shown in FIG. 3-1A. Each pixel 3-112 includes asample well 3-108 and a sensor 3-110. The sensor 3-110 may be alignedand positioned to the sample well 3-112. When an excitation source iscoupled to the integrated device, excitation energy is provided to oneor more pixels. FIG. 3-1B is a schematic illustrating coupling anexcitation source 3-106 to integrated device 3-102. Excitation source3-106 provides excitation energy 3-130 (shown in dashed lines) in theintegrated device 3-102. FIG. 3-1B illustrates the path of excitationenergy from excitation energy source 3-106 to a sample well 3-108 inpixel 3-112. Components located off of the integrated device may be usedto position and align the excitation source 3-106 to the integrateddevice. Such components may include optical components including lenses,mirrors, prisms, apertures, attenuators, and/or optical fibers.Additional mechanical components may be included in the instrumentconfigured to allow control of one or more alignment components. Suchmechanical components may include actuators, stepper motors, and/orknobs. The integrated device includes components that direct theexcitation energy 3-130 towards pixels in the integrated device. Withineach pixel 3-112, excitation energy is coupled to the sample well 3-108associated with the pixel. Although FIG. 3-1B illustrates excitationenergy coupling to each sample well in a row of pixels, in someembodiments, excitation energy may not couple to all of the pixels in arow. In some embodiments, excitation energy may couple to a portion ofpixels or sample wells in a row of pixels of the integrated device.Excitation energy may illuminate a sample located within a sample well.The sample may reach an excited state in response to being illuminatedby the excitation energy. When a sample is in an excited state, thesample may emit emission energy and the emission energy may be detectedby a sensor. FIG. 3-1B schematically illustrates the path of emissionenergy 3-140 (shown as solid lines) from the sample well 3-108 to thesensor 3-110 of a pixel 3-112. The sensor 3-110 in a pixel 3-112 may beconfigured and positioned to detect emission energy from sample well3-108. In some embodiments, the sensor 3-110 may include one or moresub-sensors.

A sample to be analyzed may be introduced into a sample well 3-108 of apixel 3-112. The sample may be a biological sample or any other suitablesample, such as a chemical sample. The sample may include multiplemolecules and the sample well may be configured to isolate a singlemolecule. In some instances, the dimensions of the sample well may actto confine a single molecule within the sample well, allowingmeasurements to be performed on the single molecule. An excitationsource 3-106 may be configured to deliver excitation energy into thesample well 3-108, so as to excite the sample or at least oneluminescent marker attached to the sample or otherwise associated withthe sample while it is within an excitation area within the sample well3-108. In a sample of multiple molecules, a type of luminescent markermay uniquely associate with a molecule type. During or after excitation,the luminescent marker may emit emission energy. When multiple markersare used, they may emit at different characteristic energies. In someembodiments, multiple markers may have different characteristiclifetimes. Additionally, multiple markers may differ in response tomultiple excitation energies. Markers for a sample may be discerned bytheir excitation response, characteristic energies or wavelengths,and/or characteristic lifetimes. Emissions from the sample may radiatefrom the sample well 3-108 to the sensor 3-110.

Components may focus emission energy towards the sensor and mayadditionally or alternatively spatially separate emission energies thathave characteristic energies or wavelengths. Excitation energy emittedfrom the excitation source 3-106 may be directed toward the sample well3-108 in any suitable way and configured to excite at least one samplethat is received in the sample well 3-108. According to someembodiments, the excitation source 3-106 may excite the sample, whichmay luminesce. The excitation source may provide one or more excitationenergies to sample well 3-108. In some implementations, the excitationsource 3-106 may excite one or more markers that luminesce or emitenergy in response to the excitation and that are attached to the sampleor otherwise associated with the sample. Emitted luminescent light orenergy resulting from the excitation may be directed to a sensor 3-110,which may be configured to detect the intensity and/or timing of thereceived emission. Non-limiting examples of luminescence arephotoluminescence, fluorescence and phosphorescence.

In some embodiments, the integrated device may include components thatdirect emission energy into a radiation pattern that is dependent on thespectral range of the emission energy. The sensor or sensor regioncontaining multiple sub-sensors may detect a spatial distribution of theemission energy that depends on the radiation pattern. Markers that emitdifferent emission energies and/or spectral ranges may form differentradiation patterns. The sensor or sensor region may detect informationabout the spatial distribution of the emission energy that can be usedto identify a marker among the multiple markers.

The emission energy or energies may be detected by the sensor andconverted to at least one electrical signal. The electrical signals maybe transmitted along conducting lines in the circuitry of the integrateddevice connected to the instrument through the integrated deviceinterface, such as integrated device interface 2-114 of instrument 2-104shown in FIG. 2-1B. The electrical signals may be subsequently processedand/or analyzed. Processing or analyzing of electrical signals may occuron a suitable computing device either located on the instrument 2-104 oroff instrument, such as computing device 2-120 shown in FIG. 2-1B.

Integrated device 2-210 may appear as depicted in FIG. 2-2. Electronic,optical, and related structures may all be incorporated onto a singlesubstrate 2-200. The integrated device may include an array of pixels2-205 and integrated electronic circuitry. The integrated electroniccircuitry may include drive and read-out circuitry 2-215 coupled to thesensors of the pixel array, and signal processing circuitry. The signalprocessing circuitry may include analog-to-digital converters 2-217 andone or more field-programmable gate arrays and/or digital signalprocessors 2-219. Some embodiments may have more circuit components, andsome embodiments may have fewer circuit components integrated on thesubstrate. Although the components of the integrated device are depictedon a single level in FIG. 2-2, the components may be fabricated onmultiple levels on the substrate 2-200.

In some embodiments, there may be optical elements (not shown) locatedon the integrated device that are arranged for guiding and couplingexcitation energy from one or more excitation sources to the samplewells. Such source-to-well elements may include plasmonic structures andother microfabricated structures located adjacent the sample wells.Additionally, in some embodiments, there may be optical elements locatedon the integrated device that are configured for guiding emission energyfrom the sample wells to corresponding sensors. Such well-to-sampleelements may include may include plasmonic structures and othermicrofabricated structures located adjacent the sample wells. In someembodiments, a single component may play a role in both in couplingexcitation energy to a sample well and delivering emission energy fromthe sample well to a corresponding sensor.

In some implementations, an integrated device may include more than onetype of excitation source that is used to excite samples at a samplewell. For example, there may be multiple excitation sources configuredto produce multiple excitation energies or wavelengths for exciting asample. In some embodiments, a single excitation source may beconfigured to emit multiple wavelengths that are used to excite samplesin the sample wells. In some embodiments, each sensor at a pixel of theintegrated device may include multiple sub-sensors configured to detectdifferent emission energy characteristics from the sample.

In operation, parallel analyses of samples within the sample wells arecarried out by exciting the samples within the wells using theexcitation source and detecting signals from sample emission with thesensors. Emission energy from a sample may be detected by acorresponding sensor and converted to at least one electrical signal.The resulting signal, or signals, may be processed on the integrateddevice in some embodiments, or transmitted to the instrument forprocessing by the processing device and/or computing device. Signalsfrom a sample well may be received and processed independently fromsignals associated with the other pixels.

When an excitation source delivers excitation energy to a sample well,at least one sample within the well may luminesce, and the resultingemission may be detected by a sensor. As used herein, the phrases “asample may luminesce” or “a sample may emit radiation” or “emission froma sample” mean that a luminescent tag, marker, or reporter, the sampleitself, or a reaction product associated with the sample may produce theemitted radiation.

In some embodiments, samples may be labeled with one or more markers,and emission associated with the markers is discernable by theinstrument. For example the sensor may be configured to convert photonsfrom the emission energy into electrons to form an electrical signalthat may be used to discern a lifetime that is dependent on the emissionenergy from a specific marker. By using markers with different lifetimesto label samples, specific samples may be identified based on theresulting electrical signal detected by the sensor. In some embodiments,components of the integrated device may affect the emission from asample well to produce a spatial emission distribution pattern that isdependent on the emission wavelength. A corresponding sensor for thesample well may be configured to detect the spatial distributionpatterns from a sample well and produce signals that differentiatebetween the different emission wavelengths, as described in furtherdetail below.

II. Integrated Device

The integrated device may be configured to receive excitation energyfrom an external excitation energy source. In some embodiments, a regionof the device may be used to couple to an excitation energy sourcelocated off the integrated device. Components of the integrated devicemay guide excitation energy from the excitation source coupling regionto at least one pixel. In some embodiments, at least one waveguide maybe configured to deliver excitation energy to at least one pixel havinga sample well. A sample located within the sample well may emit emissionenergy in response to being illuminated with excitation energy. One ormore sensors located within the pixel are configured to receive theemission energy.

Components and/or layers of integrated device 3-200 according to someembodiments shown in FIG. 3-2 include a sample well 3-203, waveguide3-220, and sensor 3-275 integrated into one device. Sample well 3-203may be formed in sample well layer 3-201 of integrated device 3-200. Insome embodiments, the sample well layer 3-201 may be metal. Sample well3-203 may have a dimension D_(tv) which may indicate a cross-sectionaldimension of the sample well. Sample well 3-203 may act as ananoaperture and have one or more sub-wavelength dimensions that createa field enhancement effect that increases the intensity of theexcitation of the sample in sample well 3-203. Waveguide 3-220 isconfigured to deliver excitation energy from excitation source 3-230located off device 3-200 to sample well 3-203. The waveguide 3-220 maybe formed in a layer between sample well layer 3-201 and sensor 3-275.The design of integrated device 3-200 allows for sensor 3-275 to collectluminescence emitted from a sample in sample well 3-203. At least someof the time, the sample absorbs excitation energy and emits a photonwith an energy less than that of the excitation energy, referred to asemission energy or luminescence.

Having sample well 3-203 and sensor 3-275 on integrated device 3-200 mayreduce the optical distance that light travels from the sample well3-203 to sensor 3-215. Dimensions of integrated device 3-200 orcomponents within the device may be configured for a certain opticaldistance. Optical properties of the materials of components and/or oneor more layers of the device may determine an optical distance between asample well and a sensor. In some embodiments, the thicknesses of one ormore layers may determine the optical distance between the sample welland sensor in a pixel. Additionally or alternatively, the index ofrefraction of materials that form one or more layers of integrateddevice 3-200 may determine the optical distance between sample well3-203 and sensor 3-275 in a pixel. Such an optical distance between thesample well and sensor in a pixel may be less than 1 mm, less than 100microns, less than 25 microns, and/or less than 10 microns. One or morelayers may be present between sample well layer 3-201 and waveguidelayer 3-220 to improve coupling of excitation energy from waveguide3-220 to sample well 3-203. Although integrated device 3-200 shown inFIG. 3-2 illustrates only a single layer 3-210, multiple layers may beformed between sample well 3-203 and waveguide 3-220. Layer 3-210 may beformed with optical properties to improve coupling of excitation energyfrom waveguide 3-220 to sample well 3-203. Layer 3-210 may be configuredto reduce scattering and/or absorption of excitation energy and/orincrease luminescence from a sample in sample well 3-203. Layer 3-210may be optically transparent, according to some embodiments, so thatlight may travel to and from the sample well 3-203 with littleattenuation. In some embodiments, dielectric materials may be used toform layer 3-210. In some embodiments, excitation energy couplingcomponents within layer 3-210 and/or at the interface between layer3-210 and sample well layer 3-201 may be provided to improve coupling ofexcitation energy from waveguide 3-220 to sample well 3-203. As anexample, energy-collection components 3-215 formed at the interfacebetween sample well layer 3-201 and layer 3-210 may be configured toimprove coupling of excitation energy from waveguide 3-220 to samplewell 3-203. Energy-collection components 3-215 are optional and, in someembodiments, the configuration of waveguide 3-220 and sample well 3-203may allow for adequate coupling excitation energy without the presenceof excitation energy collection components 3-215.

Luminescent light or energy emitted from a sample in the sample well3-203 may be transmitted to the sensor 3-275 in a variety of ways, someexamples of which are described in detail below. Some embodiments mayuse optical components to increase the likelihood that light of aparticular wavelength is directed to an area or portion of the sensor3-275 that is dedicated to detecting light of that particularwavelength. The sensor 3-275 may include multiple portions for detectingsimultaneously light of different wavelengths that may correspond toemissions from different luminescent markers.

One or more layers may be present between sample well 3-203 and sensor3-275 that may be configured to improve collection of luminescence fromsample well 3-203 to sensor 3-275. Luminescence directing components maybe located at the interface between sample well layer 3-201 and layer3-210. The energy-collection components 3-215 may focus emission energytoward the sensor 3-275, and may additionally or alternatively spatiallyseparate emission energies that have different characteristic energiesor wavelengths. Such energy-collection components 3-215 may include agrating structure for directing luminescence towards sensor 3-275. Insome embodiments, the grating structure may be a series of concentricrings or “bullseye” grating structure configuration. The concentriccircular gratings may protrude from a bottom surface of the sample welllayer 3-201. The circular gratings may act as plasmonic elements whichmay be used to decrease the spread of the signal light and direct thesignal light towards associated sensor 3-275. Such a bullseye gratingmay direct luminescence more efficiently towards sensor 3-275.

Layer 3-225 may be formed adjacent to the waveguide. The opticalproperties of layer 3-225 may be selected to improve collection ofluminescence from the sample well to sensor 3-275. In some embodiments,layer 3-225 may be a dielectric material. A baffle may be formed betweensample well layer 3-201 and sensor 3-275. Baffle 3-240 may be configuredsuch that the sensor 3-275 receives luminescence corresponding to samplewell 3-203 and reduces luminescence, and reflected/scattered excitationfrom other sample wells. Filtering elements 3-260 may be positioned andconfigured to reduce excitation energy from reaching sensor 3-275. Insome embodiments, filtering elements 3-260 may include a filter thatselectively transmits emission energy of one or more markers used tolabel a sample. In embodiments with an array of sample wells and anarray of sensors where each sample well has a corresponding sensor, abaffle corresponding to each sample well may be formed to reduceluminescence from other sample wells and reflected and/or scatteredexcitation light from being collected by a sensor corresponding to thesample well.

One or more layers may be formed between waveguide 3-220 and sensor3-275 to reduce transmission of excitation energy to sensor. In someembodiments, filtering elements may be formed between waveguide 3-220and sensor 3-275. Such filtering elements may be configured to reducetransmission of excitation energy to sensor 3-275 while allowingluminescence from the sample well to be collected by sensor 3-275.

The emission energy or energies may be detected by the sensor 3-275 andconverted to at least one electrical signal. The electrical signal orsignals may be transmitted along one or more row or column conductinglines (not shown) to the integrated electronic circuitry on thesubstrate 3-200 for subsequent signal processing.

The above description of FIG. 3-2 is an overview of some of thecomponents of the apparatus according to some embodiments. In someembodiments, one or more elements of FIG. 3-2 may be absent or in adifferent location. The components of the integrated device 3-200 andexcitation source 3-230 are described in more detail below.

A. Excitation Source Coupling Region

The integrated device may have an excitation source coupling regionconfigured to couple with an external excitation energy source and guideexcitation towards at least one pixel in a pixel area of the integrateddevice. The excitation source coupling region may include one or morestructures configured to couple light into at least one waveguide. Anysuitable mechanism for coupling excitation energy into a waveguide maybe used.

In some embodiments, excitation energy from an external excitationsource may couple to a waveguide of an integrated device throughedge-coupling. An edge of the integrated device may include an end ofthe waveguide such that an external excitation source positionedproximate to the end of the waveguide may couple light into thewaveguide. In such embodiments, fabrication of the excitation sourcecoupling region may include positioning of an end of a waveguide at anedge of integrated device. FIG. 4-1A illustrates an example ofedge-coupling. Optical fiber 4-106, configured to propagate excitationenergy, is positioned proximate to an edge of integrated device 4-102where an end of waveguide 4-104 of integrated device 4-102 is located atthe edge such that optical fiber 4-106 may couple light into waveguide4-104. In such embodiments, monitoring the alignment of optical fiber4-106 or other excitation source to waveguide 4-104 can improve theamount of light provided by the optical fiber to the waveguide.

In some embodiments, a prism may couple light to the waveguide. Lightmay be directed and refracted by the prism in order to match the opticalphase frequency of the propagating waveguide mode. The refractive indexof the material used for the prism may be selected to improve couplingwith the waveguide. In some instances, the prism has a high refractiveindex and a narrow gap relative to the waveguide. In other embodiments,light may couple directly to an end of a waveguide. An edge of theintegrated device may be sufficiently well polished to allow focusingand alignment of the light to the waveguide.

The excitation source coupling region of an integrated device mayinclude structural components configured to couple with an externalexcitation source. An integrated device may include a grating couplerconfigured to couple with an external excitation source positionedproximate a surface of the integrated device and direct light towards atleast one waveguide of the integrated device. Features of the gratingcoupler, such as the size, shape, and/or grating configurations may beformed to improve coupling of the excitation energy from the excitationsource to the waveguide. The grating coupler may include one or morestructural components where the spacing between the structuralcomponents may act to propagate light. One or more dimensions of thegrating coupler may provide desirable coupling of light having a certaincharacteristic wavelength.

An integrated device may also include a waveguide having a taperedregion at an end of the waveguide. One or more dimensions of thewaveguide perpendicular to a direction of light propagation in thewaveguide may be larger at an end of the waveguide, forming a taperedregion of the waveguide. In some embodiments, the tapered region of awaveguide may have a dimension perpendicular to the propagation of lightand parallel to a surface of the integrated device that is larger at anend of the waveguide and becomes smaller along the length of thewaveguide. In embodiments that include a grating coupler, the taperedregion can be positioned proximate to the grating coupler such that thelarger end of the tapered region is located closest to the gratingcoupler. The tapered region may be sized and shaped to improve couplingof light between the grating coupler and the waveguide by expanding oneor more dimensions of the waveguide to allow for improved mode overlapof the waveguide with the grating coupler. In this manner, an excitationsource positioned proximate the surface of an integrated device maycouple light into the waveguide via the grating coupler. Such acombination of grating coupler and waveguide taper may allow for moretolerance in the alignment and positioning of the excitation source tothe integrated device.

An exemplary integrated device having a grating coupler and a waveguidewith a tapered region is shown in FIG. 4-1B. Integrated device 4-100 hasan excitation source coupling region that includes a waveguide with atapered region 4-114 and a grating coupler 4-116. Tapered region 4-114has an end with a larger dimension parallel to surface 4-112 ofintegrated device 4-100 and perpendicular to propagation of light alongthe waveguide. The end of tapered region 4-114 may be sized and shapedto provide suitable coupling between the grating coupler 4-116 and thewaveguide. Optical fiber 4-120, or other suitable excitation source,positioned with respect to grating coupler 4-116 may couple excitationenergy to the waveguide.

A grating coupler may be positioned in a region of the integrated deviceexternal to the pixels of the integrated device. On a surface of theintegrated device, the sample wells of the pixels may occupy a region ofthe surface separate from the excitation source coupling region. Anexcitation source positioned proximate to the surface of the excitationsource coupling region may couple with the grating coupler. The samplewells may be positioned separate from the excitation source couplingregion to reduce interference of light from the excitation source onperformance of the pixels. A grating coupler of an integrated device maybe formed within one or more layers of the integrated device thatinclude a waveguide. In this manner, an excitation source couplingregion of an integrated device may include a grating coupler within thesame plane of the integrated device as a waveguide. The grating couplermay be configured for a particular set of beam parameters, includingbeam width, angle of incidence, and/or polarization of the incidentexcitation energy.

A cross-sectional view of integrated device 4-200 is shown in FIG. 4-2.Integrated device 4-200 includes at least one sample well 4-222 formedin layer 4-223 of integrated device 4-200. Integrated device 4-200includes grating coupler 4-216 and waveguide 4-220 formed insubstantially the same plane of integrated device 4-200. In someembodiments, grating coupler 4-216 and waveguide 4-220 are formed fromthe same layer of integrated device 4-200 and may include the samematerial. Excitation source coupling region 4-201 of integrated device4-200 includes grating coupler 4-216. As shown in FIG. 4-2, sample well4-222 is positioned on a surface of integrated device 4-200 external toexcitation source coupling region 4-201. Excitation source 4-214positioned relative to integrated device 4-200 may provide excitationenergy incident on surface 4-215 of integrated device 4-200 withinexcitation source coupling region 4-201. By positioning grating coupler4-216 within excitation source coupling region 4-201, grating coupler4-216 may couple with the excitation energy from excitation source 4-214and couple excitation energy to waveguide 4-220. Waveguide 4-220 isconfigured to propagate excitation energy to the proximity of one ormore sample wells 4-222.

A grating coupler may be formed from one or more materials. In someembodiments, a grating coupler may include alternating regions ofdifferent materials along a direction parallel to propagation of lightin the waveguide. As shown in FIG. 4-2, grating coupler 4-216 includesstructures that are surrounded by material 4-224. The one or morematerials that form a grating coupler may have one or more indices ofrefraction suitable for coupling and propagating light. In someembodiments, a grating coupler may include structures formed from onematerial surrounded by a material having a larger index of refraction.As an example, a grating coupler may include structures formed ofsilicon nitride and surrounded by silicon dioxide.

Any suitable dimensions and/or inter-grating spacing may be used to forma grating coupler. Grating coupler 4-216 may have a dimensionperpendicular to the propagation of light through the waveguide, such asalong the y-direction as shown in FIG. 4-2, of approximately 50 nm,approximately 100 nm, approximately 150 nm, or approximately 200 nm.Spacing between structures of the grating coupler along a directionparallel to light propagation in the waveguide, such as along thez-direction as shown in FIG. 4-2, may have any suitable distance. Theinter-spacing grating may be approximately 300 nm, approximately, 350nm, approximately, 400 nm, approximately 420 nm, approximately 450 nm,or approximately 500 nm. In some embodiments, the inter-grating spacingmay be variable within a grating coupler. Grating coupler 4-216 may haveone or more dimensions substantially parallel to surface 4-215 ofintegrated device 4-200 that provide a suitable area for coupling withexternal excitation source 4-214. The area of grating coupler 4-216 maycoincide with one or more dimensions of a beam of light from excitationsource 4-214 such that the beam overlaps with grating coupler 4-215. Agrating coupler may have an area configured for a beam diameter ofapproximately 10 microns, approximately 20 microns, approximately 30microns, or approximately 40 microns.

A cross-sectional view of a portion of an exemplary excitation sourcecoupling region of an integrated device is shown in FIG. 4-3A.Excitation source coupling region includes grating coupler 4-326 andreflection layer 4-336 configured to reflect excitation light thatpasses through grating coupler 4-326 back towards grating coupler 4-326.Grating coupler 4-326 may include structures that have an inter-gratingspacing represented by A along the z-direction shown in FIG. 4-3A. Thestructures may have a linear, curved, or any other suitable shape. Insome embodiments, grating coupler 4-326 may have a dimension along they-direction similar to waveguide 4-330, as represented by the arrows oneither side of waveguide 4-330. Grating coupler 4-326 is surrounded byregion 4-324, and the combination of materials that form grating coupler4-324 and region 4-324 may provide desired coupling of light towaveguide 4-330. The index of refraction of waveguide 4-330, gratingcoupler 4-326, and/or surrounding material 4-324 may influence thecoupling of the excitation energy to the waveguide and overall couplingefficiency of excitation energy to waveguide 4-330. An excitation sourcecoupling region of an integrated device may have more than approximately50% coupling efficiency. Grating coupler 4-326 may be configured for oneor more characteristics of incident beam of excitation energy 4-314,including a characteristic wavelength, beam diameter (represented byarrows), and beam incident angle (represented by θ). Grating coupler4-326 may be configured for a beam of light having a beam diameter ofapproximately 10 microns, approximately 20 microns, approximately 30microns, or approximately 40 microns. Grating coupler 4-326 may beconfigured for a beam of light having an incident angle of approximately2 degrees, approximately 5 degrees, or approximately 7 degrees. Gratingcoupler 4-326 may be configured to couple with excitation energy of acertain polarization, such as TM or TE polarized light.

A simulation of light coupling from a beam into a waveguide via agrating coupler is shown FIG. 4-3B. Waveguide and grating coupler liealong the z-axis at approximately the 0 point of the y-axis. Light beamis an incident angle of approximately 5 degrees from the y-direction andhas a diameter of 20 microns. The waveguide used in this simulation hasa height (along the y-direction) of 100 nm, and the rating coupler hasan inter-grating spacing of 420 nm. The waveguide and the gratingcoupler structure have an index of refraction of approximately 1.87. Thematerial surrounding the waveguide and grating structure has an index ofrefraction of approximately 1.45. FIG. 4-3B illustrates light intensityof beam coupled to grating coupler and waveguide and shows the modes oflight in the waveguide by the darker regions.

One or more dimensions of the tapered region of a waveguide and relativepositioning of the tapered region to the grating coupler may providesufficient coupling of excitation energy into the waveguide. Thecurvature and chirp of the tapered region may accommodate convergenceand/or divergence of the propagation of incident excitation energy tothe waveguide. A planar view of an exemplary waveguide layer thatincludes a grating coupler 4-316 and tapered waveguide region 4-318 isshown in FIG. 4-3C. Tapered waveguide region 4-318 has a dimensionperpendicular to the propagation of light and in the plane of FIG. 4-3Cthat gradually decreases from right to left to achieve a dimension ofwaveguide 4-320. Grating coupler 4-316 may have an area within the planeof FIG. 4-3C suitable for coupling with an external excitation source.Alignment of a beam of excitation energy to grating coupler 4-316 suchthat the beam substantially overlaps with the area of grating coupler4-316 may improve coupling of excitation energy into waveguide 4-320.The arrangement of tapered region 4-318 relative to grating coupler4-316 may provide suitable coupling efficiency. The angle of the taperedregion 4-318 and grating coupler 4-316 may be selected to improveefficiency of coupling excitation energy from the excitation source towaveguide 4-320 by reducing loss of excitation energy as the dimensionof the waveguide perpendicular to light propagation decreases.

An integrated device may include a layer formed on a side of a gratingcoupler opposite to the excitation source configured reflect light. Thelayer may reflect excitation energy that passes through the gratingcoupler towards the grating coupler. By including the layer in anintegrated device, coupling efficiency of the excitation energy to thewaveguide may be improved. An example of a reflective layer is layer4-218 of integrated device 4-200 shown in FIG. 4-2 and layer 4-336 shownin FIG. 4-3A. Layer 4-218 is positioned within excitation sourcecoupling region 4-201 of integrated device 4-200 and is configured toreflect light towards grating coupler 4-216. Layer 4-218 is formedproximate to the side of grating coupler 4-216 opposite to the incidentexcitation energy from excitation source 4-214. Positioning layer 4-218external to pixels of integrated device 4-200 may reduce interference oflayer 4-218 on the performance capabilities of the pixels. Layer may4-218 may include any suitable material. Layer 4-218 may besubstantially reflective for one or more excitation energies. In someembodiments, this layer may include Al, AlCu, and/or TiN.

B. Waveguide

An integrated device may include one or more waveguides arranged todeliver a desired amount of excitation energy to one or more samplewells of the integrated device. A waveguide positioned in the vicinityof one or more sample wells such that as excitation energy propagatesalong the waveguide a portion of excitation energy couples to the one ormore sample wells. A waveguide may couple excitation energy to aplurality of pixels and act as a bus waveguide. For example, a singlewaveguide may deliver excitation energy to a row or a column of pixelsof an integrated device. In some embodiments, a waveguide may beconfigured to propagate excitation energy having a plurality ofcharacteristic wavelengths. A pixel of an integrated device may includeadditional structures (e.g., microcavity) configured to directexcitation energy from the waveguide toward the vicinity of the samplewell. In some embodiments, a waveguide may carry an optical mode havingan evanescent tail configured to extend into a sample well and/or in aregion in the vicinity of the sample well. Additional energy-couplingstructures located near the sample well may couple energy from theevanescent tail into the sample well.

One or more dimensions of a waveguide of an integrated device mayprovide desired propagation of excitation energy along the waveguideand/or into one or more sample wells. A waveguide may have a dimensionperpendicular to the propagation of light and parallel to a plane of thewaveguide, which may be considered as a cross-sectional width. Awaveguide may have a cross-sectional width of approximately 0.4 microns,approximately 0.5 microns, approximately 0.6 microns, approximately 0.65microns, approximately 0.8 microns, approximately 1 micron, orapproximately 1.2 microns. A waveguide may have a dimensionperpendicular to the propagation of light and perpendicular to a planeof the waveguide, which may be considered as a cross-sectional height. Awaveguide may have a cross-sectional height of approximately 0.05microns, approximately 0.1 microns, approximately 0.15 microns,approximately 0.16 microns, approximately 0.17 microns, approximately0.2 microns, or approximately 0.3 microns. In some embodiments, awaveguide has a larger cross-sectional width than cross-sectionalheight. A waveguide may be positioned a certain distance from one ormore sample wells within in integrated device, such as distance D shownin FIG. 4-2 between sample well 4-222 and waveguide 4-220, that isapproximately 0.3 microns, 0.5 microns, or approximately 0.7 microns.

In an exemplary embodiment, a waveguide may have a cross-sectional widthof approximately 0.5 μm and a cross-sectional height of approximately0.1 μm, and be positioned approximately 0.5 μm below the sample welllayer. In another exemplary embodiment, a waveguide may have across-sectional width of approximately 1 μm and a cross-sectional heightof 0.18 μm, and be positioned 0.3 μm below the sample well layer.

A waveguide may be dimensioned to support a single transverse radiationmode or may be dimensioned to support multi-transverse radiation modes.In some embodiments, one or more dimensions of a waveguide may act suchthat the waveguide sustains only a single transverse mode and mayselectively propagate TE or TM polarization modes. In someimplementations, a waveguide may have highly reflective sections formedon its ends, so that it supports a longitudinal standing mode within thewaveguide. By supporting one mode, the waveguide may have reduced modalinterference effects from cross coupling of modes having differentpropagation constants. In some embodiments, the highly reflectivesections comprise a single, highly reflective surface. In otherembodiments, the highly reflective sections comprise multiple reflectivestructures that, in aggregate, result in a high reflectance. Waveguidesmay be configured to split excitation energy from a single excitationsource having a higher output intensity using waveguide beam splittersto create a plurality of excitation energy beams from a singleexcitation source. Such beam splitters may include evanescent couplingmechanisms. Additionally or alternatively, photonic crystals may be usedin the waveguide structure to improve propagation of excitation energyand/or in the material surrounding the waveguide to reduce scattering ofexcitation energy.

The position and arrangement of the waveguide with respect to othercomponents in a pixel of the integrated devices may be configured toimprove coupling of excitation energy towards a sample well, improvecollection of emission energy by the sensor, and/or reduce signal noiseintroduced by excitation energy. A waveguide may be sized and/orpositioned relative to a sample well so as to reduce interference ofexcitation energy propagating in the waveguide with emission energyemitted from the sample well. Positioning and arrangement of a waveguidein an integrated device may depend on the refractive indices of thewaveguide and material surrounding the waveguide. For example, adimension of the waveguide perpendicular to a direction of lightpropagation along the waveguide and within a plane of the waveguide maybe increased so that a substantial amount of emission energy from asample well passes through the waveguide as it propagates to the sensorof the pixel. In some implementations, a distance between a sample welland waveguide and/or waveguide thickness may be selected to reducereflections from one or more interfaces between the waveguide and asurrounding material. According to some embodiments, reflection ofemission energy by a waveguide may be reduced to less than about 5% insome embodiments, less than about 2% in some embodiments, and yet lessthan about 1% in some embodiments.

The capability of a waveguide to propagate excitation energy may dependboth on the material for the waveguide and material surrounding thewaveguide. In this manner, the waveguide structure may include a corematerial, such as waveguide 4-220, and a cladding material, such asregion 4-224 as illustrated in FIG. 4-2. The material of both thewaveguide and surrounding material may allow for propagation ofexcitation energy having a characteristic wavelength through thewaveguide. Material for either the waveguide or the surrounding materialmay be selected for particular indices of refraction or combination ofindices of refraction. The waveguide material may have a lowerrefractive index than the waveguide surrounding material. Examplewaveguide materials include silicon nitride (Si_(x)N_(y)), siliconoxynitride, silicon carbide, tantalum oxide (TaO₂), aluminum dioxide.Example waveguide surrounding materials include silicon dioxide (SiO₂)and silicon oxide. The waveguide and/or the surrounding material mayinclude one or more materials. In some instances, a desired refractiveindex for a waveguide and/or surrounding material can be obtained byforming the waveguide and/or surrounding material to include more thanone material. In some embodiments, a waveguide comprises silicon nitrideand a surrounding material comprises silicon dioxide.

In an exemplary embodiment, a waveguide comprises silicon nitride andhas a refractive index of approximately 1.90 and a cross-sectionalheight of approximately 100 nm, and the surrounding material comprisessilicon dioxide and has a refractive index of approximately 1.46. Insome embodiments, a waveguide may have a refractive index ofapproximately 1.88 may be used while the surrounding material has anindex of refraction of approximately 1.46. In such embodiments, thelower refractive index for the waveguide may reduce optical loss. Inanother exemplary embodiment, a waveguide comprises a silicon nitridecore and a silicon dioxide cladding and is configured to propagateexcitation energy having a characteristic wavelength of 635 nm. The coremay have an index of refraction of 1.99 and dimensions of 100 nm by 500nm.

Waveguides of an integrated device may be formed to have a desired levelof uniformity within a waveguide and/or among multiple waveguide.Uniformity within an integrated device can be achieved by fabricatingthe core and/cladding of a waveguide structure with substantiallysimilar dimensions and/or indices of refraction along one waveguide andamong multiple waveguides. Additionally, waveguides may be formed in arepeatable manner across different integrated devices by ensuring arepeatable fabrication process to achieve a certain level of complianceacross different devices. Variation in the cross-sectional height of awaveguide and/or multiple waveguides may be less than approximately 2%,less than approximately 3%, or less than approximately 4%. Variation inthe index of refraction of a waveguide and/or multiple waveguides may beless than approximately 0.5%, less than approximately 1%, or less thanapproximately 2%. Variation in the index of refraction of thesurrounding material or cladding of a waveguide and/or multiplewaveguide may be less than approximately 0.5%, less than approximately1%, or less than approximately 2%.

A waveguide may be located between a sample well and one or more sensorsin a pixel. For example, as shown in FIG. 4-2, waveguide 4-220 ispositioned between sample well 4-222 and layer 4-230 which includes atleast one sensor. In some embodiments, sample well 4-222 may be locatedbetween the waveguide and sensor. A waveguide may be aligned, forexample, center-to-center with the sensor such that the center of thewaveguide is substantially aligned with the center of the sample well.In some embodiments, the waveguide may be displaced from acenter-to-center alignment with the sample well by a certain distance.In some embodiments, two substantially parallel waveguides may deliverexcitation energy of a same wavelength or different wavelengths to apixel, and the sample well may be located between the two waveguides. Insome embodiments, a plurality of waveguides at different levels withinthe integrated device may direct excitation energy towards the vicinityof one or more sample wells located on the integrated device.

One or more waveguides of an integrated device may include bends.Bending of waveguides may provide a desired arrangement of waveguidesand/or pixels such that a sufficient amount of excitation energy couplesto one or more sample wells of an integrated device. The design ofwaveguide bends may balance the excitation energy loss due to bendingand the spatial extent of the bends. Additionally, with proper design,bends within a waveguide may also be used to filter out part of thepropagation light. Designing a portion of a waveguide to have a certainradius of curvature may decrease light of one polarization through thefiltering provided by the curvature in the waveguide. Such bending maybe used to select for a specific polarization mode, such as TM and TE.In some embodiments, bends may be used to filter out and/or attenuatethe TM mode and retain the TE mode. FIG. 4-4 plots the loss of light dueto bending as a function of radius of curvature for a bend for waveguidehaving a cross-sectional height of 100 nm and widths of 300 nm, 400 nm,500 nm, 700 nm, and 1000 nm. As an example, to achieve a loss of atleast 0.1 dB/90 degree bend, a waveguide having a cross-sectional widthof 500 nm may have a bend radius of more than approximately 35 micronsor a waveguide having a cross-sectional width of 700 nm may have a bendradius of more than approximately 22 microns.

Splitting waveguides from a single waveguide to multiple waveguides mayallow for excitation energy to reach multiple rows or columns of samplewells of an integrated device. An excitation source may be coupled to aninput waveguide and the input waveguide may be split into multipleoutput waveguides, where each output waveguide delivers excitationenergy to a row or column of sample wells. Any suitable techniques forsplitting and/or combining waveguides may be used. Such waveguidesplitting and/or combining techniques may include a star splitter orcoupler, a Y splitter, and/or an evanescent coupler. Additionally oralternatively, a multi-mode interference splitter (MMI) may be used forsplitting and/or combining waveguides. One or more of these waveguidesplitting and/or combining techniques may be used for directingexcitation energy to a sample well.

An example star coupler 4-500 is illustrated in FIG. 4-5. Light of twodifferent wavelengths may be input to the star coupler using a singlewaveguide or two waveguides, 4-501 and 4-502, as shown in FIG. 4-5. Thesize and shape of each input waveguide may be used to separately tunethe spread of each of the excitation light beams to match at the outputwaveguides 4-504 to increase the likelihood that similar powerdistributions are obtained across the output waveguides 4-504. The starcoupler includes a free propagation region 4-503, which may beimplemented as a slab waveguide. Free propagation region 4-503 is ahighly multi-mode region that allows the input light to propagateessentially freely in the plane of the slab waveguide. The freepropagation region 4-503 may be, for example, 300-400 microns in widthfrom the first output waveguide to the last output waveguide.

The coupling of the excitation energy to the output waveguides 4-504 maybe tuned using various parameters of the star coupler, including one ormore dimensions of individual components of the star coupler. Forexample, a dimension of the transverse cross-section of each of theinput waveguides 4-501 and 4-502, a dimension of the transversecross-section of each of the output waveguides 4-504, and the distanceof each of the output waveguides 4-504 from the input waveguides may beparameters of the star coupler to tune to improve coupling of excitationenergy to the output waveguides 4-504. In some embodiments, the starcoupler may be formed such that output waveguides 4-504 havesubstantially equal power distribution relative to one another. In someembodiments, the output waveguides near the outside edge of the starcoupler may have a different size than the output waveguides near thecenter. For example, the waveguides near the edge may have a largertransverse cross-sectional area, thereby collecting more light than theoutput waveguides near the center of the star coupler, which have asmaller transverse cross-sectional area. In some embodiments, thedistance of the output waveguides from the input waveguides may vary.For example, as illustrated in FIG. 4-5, the output waveguides near theedge may have a distance from the input waveguides 4-501 and 4-502 thatis smaller than the output waveguides near the center of the starcoupler.

Star coupler 4-500 may have any suitable number of output waveguides. Insome embodiments, a single star coupler distributes excitation energy toan entire integrated device. Thus, the number of output waveguides isequal to the number of rows of pixels in the integrated device. Forexample, there may be 128 output waveguides. In other embodiments, morethan one star coupler may be used. In such an embodiment, a first starcoupler may have 64 output waveguides and a second star coupler may have64 output waveguides such that a combination of the first and secondstar couplers provides excitation energy to 128 rows of pixels of anintegrated device.

In some embodiments, rather than having an input waveguide, one or moregrating couplers may couple excitation energy directly into a freepropagation region of a star coupler having a plurality of outputwaveguides. A single grating coupler may be used to couple a pluralityof wavelengths to a free propagation region of the star coupler. To doso, light of differing wavelengths may be incident on the gratingcoupler at differing angles. In some embodiments, multiple gratingcouplers may be used to couple excitation energy of differentwavelengths. For example, a grating coupler for each excitationwavelength may be used. A grating coupler configured as part of a freepropagation region may operate similar to one of grating couplersdescribed above, but rather than coupling light into an input waveguidethat propagates light to the star coupler, the light is coupled directlyinto the slab waveguide that forms a free-propagation region, such as4-603.

Another exemplary configuration of a star coupler is shown in FIG. 4-6.Light of two wavelengths may be input to the star coupler using gratingcouplers 4-605 and 4-606 connected to waveguides 4-609 and 4-610,respectively. Grating couplers 4-605 and 4-606 and waveguides 4-609 and4-610 may be configured to reduce loss of excitation energy by reducingthe number of bends and/or selecting for bend angles that improvepropagation of excitation energy towards free-propagation region 4-607.Output waveguides 4-608 from the star-coupler may be configured in anysuitable way to provide excitation energy to a row of pixels. In theexample shown in FIG. 4-6, there are 32 output waveguides. Thetransverse cross-sectional area of the output waveguides 4-608 proximateto free-propagation region 4-607 may vary such that an output waveguidenear an end of free-propagation region 4-607 has a larger transversecross-sectional area than an output waveguide near the center offree-propagation region 4-607. For example, as shown in FIG. 4-6, outputwaveguide 4-608 a has a larger transverse cross-sectional area area thanoutput waveguide 4-608 b which is located closer to the center offree-propagation region 4-607. Such variation in transversecross-sectional areas among output waveguides 4-608 may improvedistribution of excitation energy across output waveguides 4-608, and insome embodiments, allow delivery of a substantially similar amount ofexcitation energy by each output waveguide to a row of pixels. In thismanner, multiple rows of pixels may receive a substantially similaramount of excitation energy across the rows of pixels.

The design for waveguide splitting may be selected based on theefficiency of the splitting technique for the number of outputwaveguides. If the splitting efficiency is high, then more outputwaveguides may be produced and only a single splitting step may occur.In some embodiments, a single output waveguide from a splitter maycorrespond to each row of sample wells. Some embodiments includemultiple splitting steps to achieve a sufficient number of waveguidesconfigured to deliver excitation energy to a portion of sample wells ofan integrated device. In some embodiments, a waveguide may be furthersplit into multiple waveguides using a multi-mode interference splitter(MMI). In a MMI, an input waveguide may be split to multiple outputwaveguides where one or more dimensions of the MMI may determine thenumber of output waveguides and/or an amount of excitation energydelivered to an output waveguide. For example, as shown in FIG. 4-7, MMIsplitter 4-707 is configured to provide outputs 4-708 from input 4-710.Outputs 4-708 may couple to waveguides configured to propagateexcitation energy to rows of sample wells. In some embodiments, MMIsplitter 4-707 may have 280 outputs coupled to waveguides configured topropagate excitation energy to 280 rows of sample wells. In someembodiments, a MMI splitter is configured to receive excitation energyfrom multiple inputs and direct the excitation energy to multipleoutputs. As shown in FIG. 4-7, MMI splitter 4-717 is coupled to multipleinputs 4-720 and provides multiple outputs 4-718. In such embodiments,the number of outputs 4-718 may be greater than the number of inputs4-720. In some embodiments, inputs 4-720 may provide differentexcitation energies to MMI splitter 4-717. In other embodiments,splitting of waveguides may occur in multiple splitting steps. As anexample, splitting may occur using two sets of multi-mode interferencesplitters where an output waveguide from a first MMI splitter is used asan input for a second MMI splitter. For example, as shown in FIG. 4-7,input waveguide 4-730 is split by MMI splitter 4-727 into a plurality ofoutputs including output 4-728 coupled to MMI splitter 4-737 configuredto provide outputs 4-738. In some embodiments, MMI splitter 4-727 mayprovide 35 outputs and each of the outputs is split by another MMIsplitter, such as 4-737, into eight output waveguides. Since each of theintermediate 35 waveguides is split into eight waveguides, 280waveguides are formed in this non-limiting example.

In some embodiments, multiple input waveguides may cross-couple in anMMI splitter to form output waveguides for coupling light to samplewells of the integrated device. On such an integrated device, there maybe multiple grating couplers to couple with multiple excitation sources.A cross-coupling MMI is included to couple the multiple excitationsources together and form multiple output waveguides, as shown in FIG.4-7. Each of the multiple input waveguides may originate at one of thegrating couplers to couple light from one of the excitation sources tothe MMI splitter. Such cross-coupling of multiple excitation sources mayimprove robustness of the system against excitation source degradationand/or failure. For example, if one of the multiple excitation sourcesstops producing excitation energy, then the other excitations sourcesare available to provide sufficient excitation energy for a desiredlevel of performance of the integrated device. A compensation mechanismmay also be included to compensate for a decrease in excitation energyby one or more of the multiple excitation sources by increasing theintensity provided by the remaining excitation sources that arefunctional.

Techniques for splitting and/or combining waveguides may be selected fora reduced loss of excitation energy when the waveguide is split and/orcombined, including insertion loss. Insertion loss that occurs due tosplitting and/or combining of a waveguide may be approximately less than10 percent, approximately less than 20 percent, or approximately lessthan 30 percent. Techniques for splitting waveguides may allow for anapproximate uniform splitting of excitation energy among the multipleoutput waveguides to evenly distribute the excitation energy among eachoutput waveguide. Techniques for combining waveguides may allow for anapproximately uniform relative contribution of excitation energy fromthe multiple input waveguides across the output waveguides. In someembodiments, uniformity among the output waveguides may be approximatelyless than 10 percent, approximately less than 20 percent, orapproximately less than 30 percent. Techniques used for designing thewaveguides may be selected for a certain tolerance of fabricationparameters, including waveguide cross-sectional height, cross-sectionalwidth, and/or the index of refraction of the waveguides.

One or more dimensions of a MMI splitter influences the number of outputwaveguides and/or the efficiency of the waveguide splitting and/orcombining. Designing a MMI splitter may include determining thedimensions of a MMI splitter to have a certain number of outputwaveguides and/or a certain splitting efficiency. FIG. 4-8 illustrates asimulation of the intensity profile for light within an example MMIsplitter configured to receive light from an input waveguide and directlight into eight output waveguides. In this example, the input waveguideand the output waveguides have a cross-sectional width of 500 nm, andthe dimensions of the MMI splitter are 16.35 microns in width, W, and84.28 microns in length, L. By measuring the transmitted light at eachof the output waveguides, a coupling uniformity and/or efficiency may bemeasured. For the examplary MMI splitter shown in FIG. 4-8, theintensity of the eight output waveguides may vary by approximately 0.1%.

In some embodiments, a grating coupler may be configured to direct inputlight into a plurality of output waveguides. FIG. 4-9A illustrates aslice grating coupler which may be used to couple light having one ormore wavelengths into a plurality of output waveguides 4-904. The slicegrating is a linear grating structure that is much wider than thewavelength of the light (e.g., hundreds of microns wide). It is formedfrom alternating layers of dielectric, such as silicon nitride andsilicon oxide. In some embodiments, multiple wavelengths can be coupledto the slice grating by launching the different wavelengths at the slicegrating such that they are incident at different angles. In someembodiments, the one or more beams incident on the grating coupler has aspot size 4-603 approximately the size of the grating structure itself,as illustrated in FIG. 4-9A.

FIGS. 4-9B and 4-9C illustrate an exemplary slice grating coupler whereFIG. 4-9C is a zoomed view of region 4-906 shown in FIG. 4-9B andincludes the slice grating coupler 4-903 and output waveguides 4-905.Such a configuration provides both coupling and division of input powerof different excitation wavelengths into a plurality of outputwaveguides. In the example shown in FIG. 4-9B, there are 128 outputwaveguides 4-905. In embodiments with multiple excitation wavelengths,the grating region 4-906 has a grating pitch designed to providecoupling of the multiple excitation wavelengths to the outputwaveguides. A waveguide may be powered by a single slice in the gratingregion where the width of the slice may vary to compensate for varyingintensity of the input excitation energy beam on the grating coupler.

C. Sample Well

According to some embodiments, a sample well 5-210 may be formed at oneor more pixels of an integrated device. A sample well may comprise asmall volume or region formed at a surface of a substrate 5-105 andarranged such that samples 5-101 may diffuse into and out of the samplewell from a specimen deposited on the surface of the substrate, asdepicted in FIG. 5-1 and FIG. 5-2, which illustrate a single pixel 5-100of an integrated device. In various embodiments, a sample well 5-210 maybe arranged to receive excitation energy from a waveguide 5-240. Samples5-101 that diffuse into the sample well may be retained, temporarily orpermanently, within an excitation region 5-215 of the sample well by anadherent 5-211. In the excitation region, a sample may be excited byexcitation energy (e.g., excitation radiation 5-247), and subsequentlyemit radiation that may be observed and evaluated to characterize thesample.

In further detail of operation, at least one sample 5-101 to be analyzedmay be introduced into a sample well 5-210, e.g., from a specimen (notshown) containing a fluid suspension of samples. Excitation energy froma waveguide 5-240 may excite the sample or at least one marker attachedto the sample or included in a tag associated with the sample while itis within an excitation region 5-215 within the sample well. Accordingto some embodiments, a marker may be a luminescent molecule (e.g.,fluorophore) or quantum dot. In some implementations, there may be morethan one marker that is used to analyze a sample (e.g., distinct markersand tags that are used for single-molecule genetic sequencing asdescribed in “Real-Time DNA Sequencing from Single PolymeraseMolecules,” by J. Eid, et al., Science 323, p. 133 (2009), which isincorporated by reference in its entirety). During and/or afterexcitation, the sample or marker may emit emission energy. When multiplemarkers are used, they may emit at different characteristic energiesand/or emit with different temporal characteristics including differentlifetimes. The emission energy from the sample well may radiate orotherwise travel to a sensor 5-260 where the emission energy is detectedand converted into electrical signals that can be used to characterizethe sample.

According to some embodiments, a sample well 5-210 may be a partiallyenclosed structure, as depicted in FIG. 5-2. In some implementations, asample well 5-210 comprises a sub-micron-sized hole or opening(characterized by at least one transverse dimension D_(sw)) formed in atleast one layer of material 5-230. In some cases, the hole may bereferred to as a “nanoaperture.” The transverse dimension of the samplewell may be between approximately 20 nanometers and approximately 1micron, according to some embodiments, though larger and smaller sizesmay be used in some implementations. A volume of the sample well 5-210may be between about 10⁻²¹ liters and about 10⁻¹⁵ liters, in someimplementations. A sample well may be formed as a waveguide that may, ormay not, support a propagating mode. A sample well may be formed as awaveguide that may, or may not, support a propagating mode. In someembodiments, a sample well may be formed as a zero-mode waveguide (ZMW)having a cylindrical shape (or similar shape) with a diameter (orlargest transverse dimension) D_(sw). A ZMW may be formed in a singlemetal layer as a nano-scale hole that does not support a propagatingoptical mode through the hole.

Because the sample well 5-210 has a small volume, detection ofsingle-sample events (e.g., single-molecule events) at each pixel may bepossible even though samples may be concentrated in an examined specimenat concentrations that are similar to those found in naturalenvironments. For example, micromolar concentrations of the sample maybe present in a specimen that is placed in contact with the integrateddevice, but at the pixel level only about one sample (or single moleculeevent) may be within a sample well at any given time. Statistically,some sample wells may contain no samples and some may contain more thanone sample. However, an appreciable number of sample wells may contain asingle sample (e.g., at least 30% in some embodiments), so thatsingle-molecule analysis can be carried out in parallel for a largenumber of pixels. Because single-molecule or single-sample events may beanalyzed at each pixel, the integrated device makes it possible todetect rare events that may otherwise go unnoticed in ensemble averages.

A transverse dimension D_(sw) of a sample well may be between about 500nanometers (nm) and about one micron in some embodiments, between about250 nm and about 500 nm in some embodiments, between about 100 nm andabout 250 nm in some embodiments, and yet between about 20 nm and about100 nm in some embodiments. According to some implementations, atransverse dimension of a sample well is between approximately 80 nm andapproximately 180 nm, or between approximately one-quarter andone-eighth of the excitation wavelength or emission wavelength.According to other implementations, a transverse dimension of a samplewell is between approximately 120 nm and approximately 170 nm. In someembodiments, the depth or height of the sample well 5-210 may be betweenabout 50 nm and about 500 nm. In some implementations, the depth orheight of the sample well 5-210 may be between about 80 nm and about 250nm.

A sample well 5-210 having a sub-wavelength, transverse dimension canimprove operation of a pixel 5-100 of an integrated device in at leasttwo ways. For example, excitation energy incident on the sample wellfrom a side opposite the specimen may couple into the excitation region5-215 with an exponentially decreasing power, and not propagate throughthe sample well to the specimen. As a result, excitation energy isincreased in the excitation region where it excites a sample ofinterest, and is reduced in the specimen where it would excite othersamples that would contribute to background noise. Also, emission from asample retained at a base of the well (e.g., nearer to the sensor 5-260)is preferably directed toward the sensor, since emission propagating upthrough the sample well is highly suppressed. Both of these effects canimprove signal-to-noise ratio at the pixel. The inventors haverecognized several aspects of the sample well that can be improved tofurther boost signal-to-noise levels at the pixel. These aspects relateto sample well shape and structure, and also to adjacent optical andplasmonic structures (described below) that aid in coupling excitationenergy to the sample well and emitted radiation from the sample well.

According to some embodiments, a sample well 5-210 may be formed as ananoaperture configured to not support a propagating mode for particularwavelengths of interest. In some instances, the nanoaperture isconfigured where all modes are below a threshold wavelength and theaperture may be a sub-cutoff nanoaperture (SCN). For example, the samplewell 5-210 may comprise a cylindrically-shaped hole or bore in aconductive layer. The cross-section of a sample well need not be round,and may be elliptical, square, rectangular, or polygonal in someembodiments. Excitation energy 5-247 (e.g., visible or near infraredradiation) may enter the sample well through an entrance aperture 5-212that may be defined by walls 5-214 of the sample well at a first end ofthe well, as depicted in FIG. 5-2. When formed as a SCN, the excitationenergy may decay exponentially along a length of the nanoaperture (e.g.in the direction of the specimen). In some implementations, thewaveguide may comprise a SCN for emitted radiation from the sample, butmay not be a SCN for excitation energy. For example, the aperture andwaveguide formed by the sample well may be large enough to support apropagating mode for the excitation energy, since it may have a shorterwavelength than the emitted radiation. The emission, at a longerwavelength, may be beyond a cut-off wavelength for a propagating mode inthe waveguide. According to some embodiments, the sample well 5-210 maycomprise a SCN for the excitation energy, such that the greatestintensity of excitation energy is localized to an excitation region5-215 of the sample well at an entrance to the sample well 5-210 (e.g.,localized near the interface between layer 5-235 and layer 5-230 asdepicted in the drawing). Such localization of the excitation energy canimprove localization of emission energy from the sample, and limit theobserved emission that emitted from a single sample (e.g., a singlemolecule).

According to some embodiments, a pixel 5-100 may include additionalstructures. For example, a pixel 5-100 may include one or moreexcitation-coupling structure 5-220 that affects coupling of excitationenergy to a sample within the sample well. A pixel may also include anemission-directing structure 5-250 that affects directing emissionenergy from a sample within the sample well to sensor 5-260.

An example of excitation localization near an entrance of a sample wellthat comprises a SCN is depicted in FIG. 5-3. A numerical simulation wascarried out to determine intensity of excitation radiation within andnear a sample well 5-210 formed as a SCN. The results show that theintensity of the excitation radiation is about 70% of the incidentenergy at an entrance aperture of the sample well and drops to about 20%of the incident intensity within about 100 nm in the sample well. Forthis simulation, the characteristic wavelength of the excitation energywas 633 nm and the diameter of the sample well 5-210 was 140 nm. Thesample well 5-210 was formed in a layer of gold metal. Each horizontaldivision in the graph is 50 nm. As shown by the graph, more thanone-half of the excitation energy received in the sample well islocalized to about 50 nm within the entrance aperture 5-212 of thesample well.

To improve the intensity of excitation energy that is localized at thesample well, other sample well structures were developed and studied bythe inventors. FIG. 5-4 depicts an embodiment of a sample well thatincludes a cavity or divot 5-216 at an excitation end of the samplewell. As can be seen in the simulation results of FIG. 5-3, a region ofhigher excitation intensity exists just before the entrance aperture5-212 of the sample well. Adding a divot 5-216 to the sample well allowsa sample to move into a region of higher excitation intensity, accordingto some embodiments. In some implementations, the shape and structure ofthe divot alters the local excitation field (e.g., because of adifference in refractive index between the layer 5-235 and fluid in thesample well), and can further increase the intensity of the excitationenergy in the divot. Divot 5-216 may be formed within layer 5-235 suchthat a portion of the sample volume that occupies sample well 5-214 anddivot 5-216 is surrounded by the material that forms layer 5-216.

The divot may have any suitable shape. The divot may have a transverseshape that is substantially equivalent to a transverse shape of thesample well, e.g., round, elliptical, square, rectangular, polygonal,etc. In some embodiments, the sidewalls of the divot may besubstantially straight and vertical, like the walls of the sample well.In some implementations, the sidewalls of the divot may be sloped and/orcurved, as depicted in the drawing. The transverse dimension of thedivot may be approximately the same size as the transverse dimension ofthe sample well in some embodiments, may be smaller than the transversedimension of the sample well in some embodiments, or may be larger thanthe transverse dimension of the sample well in some embodiments. Thedivot 5-216 may extend between approximately 10 nm and approximately 200nm beyond sample well layer 5-230. In some implementations, the divotmay extend between approximately 50 nm and approximately 150 nm beyondsample well layer 5-230. In some embodiments, the divot may extendbetween approximately 150 nm and approximately 250 nm beyond sample welllayer 5-230. By forming the divot, the excitation region 5-215 mayextend outside the sample well, as depicted in FIG. 5-4.

FIG. 5-5 depicts improvement of excitation energy at the excitationregion for a sample well containing a divot (shown in the leftsimulation image). For comparison, the excitation field is alsosimulated for a sample well without a divot, shown on the right. Thefield magnitude has been converted from a color rendering in theseplots, and the dark region at the base of the divot represents higherintensity than the light region within the sample well. The dark regionsabove the sample well represent the lowest intensity. As can be seen,the divot allows a sample 5-101 to move to a region of higher excitationintensity, and the divot also increases the localization of region ofhighest intensity at an excitation end of the sample well. Note that theregion of high intensity is more distributed for the sample well withoutthe divot. In some embodiments, the divot 5-216 provides an increase inexcitation energy at the excitation region by a factor of two or more.In some implementations, an increase of more than a factor of two can beobtained depending on the shape and depth of the divot. In thesesimulations, the layer containing the sample well includes aluminum andhas a thickness of approximately 100 nm, the divot has a depth ofapproximately 50 nm, and the excitation energy wavelength is 635 nm.

FIG. 5-6A depicts another embodiment of a sample well 5-210 in which thesample well is formed above a protrusion 5-615 at a surface of asubstrate. A resulting structure for the sample well may increase theexcitation energy at the sample by more than a factor of two compared toa sample well shown in FIG. 5-1, and may condense emission from thesample well to a sensor 5-260. According to some embodiments, aprotrusion 5-615 is patterned in a first layer 5-610 of material. Insome embodiments, the protrusion comprises a waveguide. The protrusionmay be formed as a ridge with a rectangular cross-section in someimplementations, and a second layer 5-620 of material may be depositedover the first layer of the protrusion. At the protrusion, the secondlayer may form a shape above the protrusion that approximates acylindrical portion 5-625, as depicted. In some embodiments, aconductive layer 5-230 (e.g., a reflective metal) may be deposited overthe second layer 5-620 and patterned to form a sample well 5-210 in theconductive layer above the protrusion. A divot 5-216 may then be etchedinto the second layer. The divot 5-216 may extend between about 50 nmand about 150 nm below the conductive layer 5-230. According to someembodiments, the first layer 5-610 and second layer 5-620 may beoptically transparent, and may or may not be formed of a same material.In some implementations, the first layer 5-610 may be formed from anoxide (e.g., SiO₂) or a nitride (e.g., Si₃N₄), and the second layer5-620 may be formed from an oxide or a nitride.

According to some embodiments, the conductive layer 5-230 above theprotrusion 5-615 is shaped approximately as a cylindrical reflector5-630. The shape of the cylindrical portion may be controlled byselection of the protrusion height h, width or transverse dimension w ofthe protrusion, and a thickness t of the second layer 5-620. Thelocation of the excitation region and position of the sample can beadjusted with respect to an optical focal point of the cylindricalreflector by selection of the divot depth d. It may be appreciated thatthe cylindrical reflector 5-630 can concentrate excitation energy at theexcitation region 5-215, and can also collect radiation emitted from asample and reflect and concentrate the radiation toward the sensor5-260.

Some embodiments relate to an integrated device having a sample wellwith a divot positioned proximate to a waveguide. FIG. 5-6B shows anintegrated device having sample well 5-632 formed in layer 5-630 andlayer 5-636. Layer 5-630 may be a metal layer and include one or moremetals (e.g., Al). Layer 5-636 may act as a dielectric layer and includeone or more dielectric materials (e.g., silicon dioxide). Sample well5-632 may have a variable dimension in a direction parallel to layer5-630 and/or layer 5-636. Sample well 5-632 may have a dimension D2along the z-direction at least within layer 5-630 of the integrateddevice, and in some embodiments, dimension D2 may be considered adiameter of sample well 5-632. Dimension D2 of sample well 5-632 may beapproximately 700 nm, approximately 800 nm, approximately 900 nm,approximately 1 micron, or approximately 1.1 microns. Sample well 5-632may have a dimension D1 along the z-direction within layer 5-636 of theintegrated device and in some embodiments, may be consider a diameter ata surface of sample well 5-632. Dimension D1 may be approximately 100nm, approximately 150 nm, approximately 200 nm, or approximately 250 nm.The surface of sample well 5-632 having dimension D1 is positioned adimension d1 along the x-direction from waveguide 5-634. Positioningsample well 5-632 proximate to waveguide 5-634 by distance d1 may allowfor improved coupling of excitation energy from waveguide 5-634 tosample well 5-632. Dimension d1 may be approximately 50 nm,approximately 100 nm, approximately 150 nm, approximately 200 nm, orapproximately 250 nm.

As noted above, a sample well may be formed in any suitable shape, andis not limited to only cylindrical shapes. In some implementations, asample well may be conic, tetrahedron, pentahedron, etc. FIG. 5-7A-FIG.5-7F illustrate some example sample well shapes and structures that maybe used in some embodiments. A sample well 5-210 may be formed to havean entrance aperture 5-212 that is larger than an exit aperture 5-218for the excitation energy, according to some embodiments. The sidewallsof the sample well may be tapered or curved. Forming a sample well inthis manner can admit more excitation energy to the excitation region,yet still appreciably attenuate excitation energy that travels towardthe specimen. Additionally, emission radiated by a sample maypreferentially radiate toward the end of the sample well with the largeraperture, because of favorable energy transfer in that direction.

In some embodiments, a divot 5-216 may have a smaller transversedimension than the base of the sample well, as depicted in FIG. 5-7B. Asmaller divot may be formed by coating sidewalls of the sample well witha sacrificial layer before etching the divot, and subsequently removingthe sacrificial layer. A smaller divot may be formed to retain a samplein a region that is more equidistant from the conductive walls of thesample well. Retaining a sample equidistant from the walls of the samplewell may reduce undesirable effects of the sample well walls on theradiating sample, e.g., quenching of emission, and/or altering ofradiation lifetimes.

FIGS. 5-7C and 5-7D depict another embodiment of a sample well.According to this embodiment, a sample well 5-210 may compriseexcitation-energy-enhancing structures 5-711 and an adherent 5-211formed adjacent the excitation-energy-enhancing structures. Theenergy-enhancing structures 5-711 may comprise surface plasmon ornano-antenna structures formed in conductive materials on an opticallytransparent layer 5-235, according to some embodiments. FIG. 5-7Cdepicts an elevation view of the sample well 5-210 and nearby structure,and FIG. 5-7D depicts a plan view. The excitation-energy-enhancingstructures 5-711 may be shaped and arranged to enhance excitation energyin a small localized region. For example, the structures may includepointed conductors having acute angles at the sample well that increasethe intensity of the excitation energy within an excitation region5-215. In the depicted example, the excitation-energy-enhancingstructures 5-711 are in the form of a bow-tie. Samples 5-101 diffusinginto the region may be retained, temporarily or permanently, by theadherent 5-211 and excited by excitation energy that may be deliveredfrom a waveguide 5-240 located adjacent the sample well 5-210. Accordingto some embodiments, the excitation energy may drive surface-plasmonwaves in the energy-enhancing structures 5-711. The resultingsurface-plasmon currents may produce high electric fields at the sharppoints of the structures 5-711, and these high fields may excite asample retained in the excitation region 5-215. In some embodiments, asample well 5-210 depicted in FIG. 5-7C may include a divot 5-216.

Another embodiment of a sample well is depicted in FIG. 5-7E, and showsan excitation-energy-enhancing structure 5-720 formed along interiorwalls of the sample well 5-210. The excitation-energy-enhancingstructure 5-720 may comprise a metal or conductor, and may be formedusing an angled (or shadow), directional deposition where the substrateon which the sample well is formed is rotated during the deposition.During the deposition, the base of the sample well 5-210 is obscured bythe upper walls of the well, so that the deposited material does notaccumulate at the base. The resulting structure 5-720 may form an acuteangle 5-722 near the bottom of the structure, and this acute angle ofthe conductor can enhance excitation energy within the sample well.

In an embodiment depicted in FIG. 5-7E, the material 5-232 in which thesample well is formed need not be a conductor, and may be any suitabledielectric. According to some implementations, the sample well 5-210 andexcitation-energy-enhancing structure 5-720 may be formed at a blindhole etched into a dielectric layer 5-235, and a separate layer 5-232need not be deposited.

In some implementations, a shadow evaporation may be subsequentlyperformed on the structure shown in FIG. 5-7E to deposit a metallic orconductive energy-enhancing structure, e.g., a trapezoidal structure orpointed cone at the base of the sample well, as depicted by the dashedline. The energy-enhancing structure may enhance the excitation energywithin the sample well via surface plasmons. After the shadowevaporation, a planarizing process (e.g., a chemical-mechanicalpolishing step or a plasma etching process) may be performed to removeor etch back the deposited material at the top of the sample well, whileleaving the energy-enhancing structure within the well.

In some embodiments, a sample well 5-210 may be formed from more than asingle metal layer. FIG. 5-7F illustrates a sample well formed in amulti-layer structure, where different materials may be used for thedifferent layers. According to some embodiments, a sample well 5-210 maybe formed in a first layer 5-232 (which may be a semiconducting orconducting material), a second layer 5-234 (which may be an insulator ordielectric), and a third layer 5-230 (which may be a conductor orsemiconductor). In some embodiments, a degeneratively-dopedsemiconductor or graphene may be used for a layer of the sample well. Insome implementations, a sample well may be formed in two layers, and inother implementations a sample well may be formed in four or morelayers. In some embodiments, multi-layer materials used for forming asample well may be selected to increase or suppress interfacial excitonswhich may be generated by excitation radiation incident on the samplewell. In some embodiments, multi-layer materials used for forming asample well may be selected to increase surface-plasmon generation at abase of the sample well or suppress surface-plasmon radiation at a topof the well. In some embodiments, multi-layer materials used for forminga sample well may be selected to suppress excitation radiation frompropagating beyond the sample well and multi-layer structure into thebulk specimen. In some embodiments, multi-layer materials used forforming a sample well may be selected to increase or suppressinterfacial excitons which may be generated by excitation radiationincident on the sample well.

Various materials may be used to form sample wells described in theforegoing embodiments. According to some embodiments, a sample well5-210 may be formed from at least one layer of material 5-230, which maycomprise any one of or a combination of a conductive material, asemiconductor, and an insulator. In some embodiments, the sample well5-210 comprises a highly conductive metallic layer, e.g., gold, silver,aluminum, copper. In some embodiments, the layer 5-230 may comprise amulti-layer stack that includes any one of or a combination of gold,silver, aluminum, copper, titanium, titanium nitride, palladium,platinum, and chromium. In some implementations, other metals may beused additionally or alternatively. According to some embodiments, asample well may comprise an alloy such as AlCu or AlSi.

In some embodiments, the multiple layers of different metals or alloysmay be used to form a sample well. In some implementations, the materialin which the sample well 5-210 is formed may comprise alternating layersof metals and non-metals, e.g., alternating layers of metal and one ormore oxides. In some embodiments, the non-metal may include a polymer,such as polyvinyl phosphonic acid or a polyethylene glycol (PEG)-thiol.

A layer 5-230 in which a sample well is formed may be deposited on oradjacent to at least one optically transparent layer 5-235, according tosome embodiments, so that excitation energy (in the form of opticalradiation, such as visible or near-infrared radiation) and emissionenergy (in the form of optical radiation, such as visible ornear-infrared radiation) may travel to and from the sample well 5-210without significant attenuation. For example, excitation energy from awaveguide 5-240 may pass through the at least one optically transparentlayer 5-235 to the excitation region 5-215, and emission from the samplemay pass through the same layer or layers to the sensor 5-260. Thisexcitation energy may be from the evanescent tail of excitation lightguided by the waveguide.

In some embodiments, at least one surface of the sample well 5-210 maybe coated with one or more layers 5-211, 5-280 of material that affectthe action of a sample within the sample well, as depicted in FIG. 5-8.For example, a thin dielectric layer 5-280 (e.g., alumina, titaniumnitride or silica) may be deposited as a passivating coating onsidewalls of the sample well. Such a coating may be implemented toreduce sample adhesion of a sample outside the excitation region 5-215,or to reduce interaction between a sample and the material 5-230 inwhich the sample well 5-210 is formed. The thickness of a passivatingcoating within the sample well may be between about 5 nm and about 50nm, according to some embodiments.

In some implementations, a material for a coating layer 5-280 may beselected based upon an affinity of a chemical agent for the material, sothat the layer 5-280 may be treated with a chemical or biologicalsubstance to further inhibit adhesion of a sample species to the layer.For example, a coating layer 5-280 may comprise alumina, which may bepassivated with a polyphosphonate passivation layer, according to someembodiments. Additional or alternative coatings and passivating agentsmay be used in some embodiments.

According to some embodiments, at least a bottom surface of the samplewell 5-210 and/or divot 5-216 may be treated with a chemical orbiological adherent 5-211 (e.g., biotin) to promote retention of asample. The sample may be retained permanently or temporarily, e.g., forat least a period of time between about 0.5 milliseconds and about 50milliseconds. In another embodiment, the adherent may promote temporaryretention of a sample 5-101 for longer periods. Any suitable adherentmay be used in various embodiments, and is not limited to biotin.

According to some embodiments, the layer of material 5-235 adjacent thesample well may be selected based upon an affinity of an adherent forthe material of that layer. In some embodiments, passivation of thesample well's sidewalls may inhibit coating of an adherent on thesidewalls, so that the adherent 5-211 preferentially deposits at thebase of the sample well. In some embodiments, an adherent coating mayextend up a portion of the sample well's sidewalls. In someimplementations, an adherent may be deposited by an anisotropic physicaldeposition process (e.g., evaporation, sputtering), such that theadherent accumulates at the base of a sample well or divot and does notappreciably form on sidewalls of the sample well.

Various fabrication techniques may be employed to fabricate sample wells5-210 for an integrated device. A few example processes are describedbelow, but the invention is not limited to only these examples.

The sample well 5-210 may be formed by any suitable micro- ornano-fabrication process, which may include, but is not limited to,processing steps associated with photolithography, deep-ultravioletphotolithography, immersion photolithography, near-field optical contactphotolithography, EUV lithography, x-ray lithography, nanoimprintlithography, interferometric lithography, step-and-flash lithography,direct-write electron beam lithography, ion beam lithography, ion beammilling, lift-off processing, reactive-ion etching, etc. According tosome embodiments, a sample well 5-210 may be formed usingphotolithography and lift-off processing. Example fabrication stepsassociated with lift-off processing of a sample well are depicted inFIG. 5-9A-F. Although fabrication of only a single sample well orstructure at a pixel is typically depicted in the drawings, it will beunderstood that a large number of sample wells or structures may befabricated on a substrate (e.g., at each pixel) in parallel.

According to some embodiments, a layer 5-235 (e.g., an oxide layer) on asubstrate may be covered with an anti-reflection coating (ARC) layer5-910 and photoresist 5-920, as depicted in FIG. 5-9A. The photoresistmay be exposed and patterned using photolithography and development ofthe resist. The resist may be developed to remove exposed portions orunexposed portions (depending on the resist type), leaving a pillar5-922 that has a diameter approximately equal to a desired diameter forthe sample well, D_(sw), as depicted in FIG. 5-9B. The height of thepillar may be substantially different than a desired depth of the samplewell. For example, the height of the pillar may be substantially greaterthan a desired depth of the sample well.

The pattern of the pillar 5-922 may be transferred to the ARC layer5-910 via anisotropic, reactive ion etching (RIE), for example as shownin FIG. 5-9C. The region may then be coated with at least one material5-230, e.g., a conductor or metal, which is desired to form the samplewell. A portion of the deposited material, or materials, forms a cap5-232 over the pillar 5-922, as depicted in FIG. 5-9D. The photoresist5-920 and ARC layer 5-910 may then be stripped from the substrate, usinga selective removal process (e.g., using a chemical bath with or withoutagitation which dissolves at least the resist and releases or “liftsoff” the cap). If the ARC layer 5-910 remains, it may be stripped fromthe substrate using a selective etch, leaving the sample well 5-210 asshown in FIG. 5-9E. According to some embodiments, the sidewalls 5-214of the sample well may be sloped due to the nature of the deposition ofthe at least one material 5-230.

As used herein, a “selective etch” means an etching process in which anetchant selectively etches one material that is desired to be removed oretched at a higher rate (e.g., at least twice the rate) than the etchantetches other materials which are not intended to be removed.

Because the photoresist 5-920 and ARC layer 5-910 are typically polymerbased, they are considered soft materials which may not be suitable forforming sample wells having high aspect ratios (e.g., aspect ratiosgreater than about 2:1 with respect to height-to-width). For samplewells having higher aspect ratios, a hard material may be included inthe lift-off process. For example, before depositing the ARC layer andphotoresist, a layer of a hard (e.g., an inorganic material) may bedeposited. In some embodiments, a layer of titanium or silicon nitridemay be deposited. The layer of hard material should exhibit preferentialetching over the material, or materials, 5-230 in which the sample wellis formed. After the photoresist is patterned, a pattern of the pillarmay be transferred into the ARC layer and the underlying hard material5-930 yielding a structure as depicted in FIG. 5-9F. The photoresist andARC layer may be then stripped, the material(s) 5-230 deposited, and alift-off step performed to form the sample well.

According to some embodiments, a lift-off process may be used to form asample well comprising energy-enhancing structures 5-711, as depicted inFIG. 5-7C and FIG. 5-7D.

An alternative process for forming a sample well is depicted in FIG.5-10A-D. In this process, the sample well may be directly etched into atleast one material 5-230. For example, at least one material 5-230 inwhich a sample well is to be formed may be deposited on a substrate. Thelayer may be covered by an ARC layer 5-910 and a photoresist 5-920, asillustrated in FIG. 5-10A. The photoresist may be patterned to form ahole having a diameter approximately equal to a desired diameter of thesample well, as depicted in FIG. 5-10B. The pattern of the hole may betransferred to the ARC and through the layer 5-230 using an anisotropic,reactive ion etch, as shown in FIG. 5-10C for example. The photoresistand ARC layer may be stripped, yielding a sample well as depicted inFIG. 5-10D. According to some embodiments, the sidewalls of a samplewell formed by etching into the layer of material 5-230 may be morevertical than sidewalls resulting from a lift-off process.

In some embodiments, the photoresist and ARC layer may be used topattern a hard mask (e.g., a silicon nitride or oxide layer, not shown)over the material 5-230. The patterned hole may then be transferred tothe hard mask, which is then used to transfer the pattern into the layerof material 5-230. A hard mask may allow greater etching depths into thelayer of material 5-230, so as to form sample wells of higher aspectratio.

It will be appreciated that the lift-off processes and the directetching fabrication techniques described above may be used to form asample well when multiple layers of different materials are used to forma stack of material 5-230 in which the sample well is formed. An examplestack is shown in FIG. 5-11. According to some embodiments, a stack ofmaterial may be used to form a sample well to improve coupling ofexcitation energy to the excitation region of a sample well, or toreduce transmission or re-radiation of excitation energy into the bulkspecimen. For example, an absorbing layer 5-942 may be deposited over afirst layer 5-940. The first layer may comprise a metal or metal alloy,and the absorbing layer may comprise a material that inhibits surfaceplasmons, e.g., amorphous silicon, TaN, TiN or Cr. In someimplementations, a surface layer 5-944 may also be deposited topassivate the surface surrounding the sample well (e.g., inhibitadhesion of molecules).

Formation of a sample well including a divot 5-216 may be done in anysuitable manner. In some embodiments, a divot may be formed by etchingfurther into an adjacent layer 5-235, and/or any intervening layer orlayers, adjacent the sample well. For example, after forming a samplewell in a layer of material 5-230, that layer 5-230 may be used as anetch mask for patterning a divot, as depicted in FIG. 5-12. For example,the substrate may be subjected to a selective, anisotropic reactive ionetch so that a divot 5-216 may be etched into adjacent layer 5-235. Forexample, in an embodiment where the material 5-230 is metallic and theadjacent layer 5-235 silicon oxide, a reactive-ion plasma etch having afeed gas comprising CHF₃ or CF₄ may be used to preferentially removeexposed silicon oxide below the sample well and form the divot 5-216. Asused herein, “silicon oxide” generally refers to SiO_(x) and may includesilicon dioxide, for example.

In some embodiments, conditions within the plasma (e.g., bias to thesubstrate and pressure) during an etch may be controlled to determinethe etch profile of the divot. For example, at low pressure (e.g., lessthan about 100 mTorr) and high DC bias (e.g., greater than about 20V),the etching may be highly anisotropic and form substantially straightand vertical sidewalls of the divot, as depicted in the drawing. Athigher pressures and lower bias, the etching may be more isotropicyielding tapered and/or curved sidewalls of the divot. In someimplementations, a wet etch may be used to form the divot, which may besubstantially isotropic and form an approximately spherical divot thatmay extend laterally under the material 5-230, up to or beyond thesidewalls of the sample well.

FIG. 5-13A through FIG. 5-13C depict process steps that may be used toform a divot 5-216 having a smaller transverse dimension than the samplewell 5-210 (for example, a divot like that depicted in FIG. 5-7B). Insome implementations, after forming a sample well, a conformalsacrificial layer 5-960 may be deposited over a region including thesample well. According to some embodiments, the sacrificial layer 5-960may be deposited by a vapor deposition process, e.g., chemical vapordeposition (CVD), plasma-enhanced CVD, or atomic layer deposition (ALD).The sacrificial layer may then be etched back using a first anisotropicetch that is selective to the sacrificial layer 5-960, removes the layerfrom horizontal surfaces, leaves side wall coatings 5-962 on walls ofthe sample well, as depicted in FIG. 5-13B. The etch back may beselective and stop on the material 5-230 and adjacent layer 5-235 insome embodiments, or may be a non-selective, timed etch in someembodiments.

A second anisotropic etch that is selective to the adjacent layer 5-235may be executed to etch a divot 5-216 into the adjacent layer asdepicted in FIG. 5-13C. The sacrificial side wall coatings 5-962 maythen optionally be removed by a selective wet or dry etch. The removalof the sidewall coatings open up the sample well to have a largertransverse dimension than the divot 5-216.

According to some embodiments, the sacrificial layer 5-960 may comprisethe same material as the adjacent layer 5-235. In such embodiments, thesecond etch may remove at least some of the side wall coating 5-962 asthe divot is etched into the adjacent layer 5-235. This etch back of theside wall coating can form tapered sidewalls of the divot in someembodiments.

In some implementations, the sacrificial layer 5-960 may be formed from,or include a layer of, a material that is used to passivate thesidewalls of the sample well (e.g., reduce adhesion of samples at thesidewalls of the sample well). At least some of the layer 5-960 may thenbe left on the walls of the sample well after formation of the divot.

According to some embodiments, the formation of the sidewall coatings5-962 occurs after the formation of the divot. In such embodiments, thelayer 5-960 coats the sidewalls of the divot. Such a process may be usedto passivate the sidewalls of the divot and localize the sample within acenter region of the divot.

Process steps associated with depositing an adherent 5-211 at a base ofa sample well 5-210, and a passivation layer 5-280 are depicted in FIG.5-14. According to some embodiments, a sample well may include a firstpassivation layer 5-280 on walls of the sample well. The firstpassivation layer may be formed, for example, as described above inconnection with FIG. 5-13B or FIG. 5-8. In some embodiments, a firstpassivation layer 5-280 may be formed by any suitable deposition processand etch back. In some embodiments, a first passivation layer may beformed by oxidizing the material 5-230 in which the sample well isformed. For example, the sample well may be formed of aluminum, whichmay be oxidized to create a coating of alumina on sidewalls of thesample well.

An adherent 5-980 or an adherent precursor (e.g., a material whichpreferentially binds an adherent) may be deposited on the substrateusing an anisotropic physical deposition process, e.g., an evaporativedeposition, as depicted in FIG. 5-14A. The adherent or adherentprecursor may form an adherent layer 5-211 at the base of the samplewell, as depicted in FIG. 5-14B, and may coat an upper surface of thematerial 5-230 in which the sample well is formed. A subsequent angled,directional deposition depicted in FIG. 5-14C (sometimes referred to asa shadow deposition or shadow evaporation process) may be used todeposit a second passivation layer 5-280 of passivation material 5-990over an upper surface of the material 5-230 without covering theadherent layer 5-211. During the shadow deposition process, thesubstrate may be rotated around an axis normal to the substrate, so thatthe second passivation layer 5-280 deposits more uniformly around anupper rim of the sample well. A resulting structure is depicted in FIG.5-14D, according to some embodiments. As an alternative to depositingthe second passivation layer, a planarizing etch (e.g., a CMP step) maybe used to remove adherent from an upper surface of the material 5-230.

According to some implementations, an adherent layer 5-211 may bedeposited centrally at the base of a tapered sample well, as depicted inFIG. 5-15. For example, an adherent, or adherent precursor, may bedirectionally deposited, as depicted in FIG. 5-14A, in a tapered samplewell, formed as described above. Walls of the sample well may bepassivated by an oxidation process before or after deposition of theadherent layer 5-211. Adherent or precursor remaining on a surface ofthe material 5-230 may be passivated as described in connection withFIG. 5-14D. In some embodiments, an adherent on an upper surface of thematerial 5-230 may be removed by a chemical-mechanical polishing step.By forming an adherent layer, or an adherent layer precursor, centrallyat the base of a sample well, deleterious effects on emission from asample (e.g., suppression or quenching of sample radiation from samplewalls, unfavorable radiation distribution from a sample because it isnot located centrally with respect to energy coupling structures formedaround a sample well, adverse effects on luminescent lifetime for asample) may be reduced.

In some embodiments, lift-off patterning, etching, and depositionprocesses used to form the sample well and divot may be compatible withCMOS processes that are used to form integrated CMOS circuits on anintegrated device. Accordingly, an integrated device may be fabricatedusing conventional CMOS facilities and fabrication techniques, thoughcustom or specialized fabrication facilities may be used in someimplementations.

Variations of the process steps described above may be used to formalternative embodiments of sample wells. For example, a tapered samplewell such as depicted in FIG. 5-7A or FIG. 5-7B may be formed using anangled deposition process depicted in FIG. 5-14C. For the sample well ofFIG. 5-7B, the angle of deposition may be changed during the depositionprocess. For such embodiments, a sample well having substantiallystraight and vertical sidewalls may first be formed, and then additionalmaterial 5-230 deposited by an angled deposition to taper the sidewallsof the sample well.

In some embodiments, a sample well may be formed from a multi-layerstack comprising a plurality of layers. FIG. 5-16 illustrates a samplewell with a divot that is formed in the substrate layer 5-105. Thesample well in this embodiment is approximately 140-180 nm in diameterwith a divot depth of approximately 40-90 nm. The substrate 5-105 may beformed from any suitable material, such as silicon oxide. A first layer5-1001 may be formed on the surface of the substrate 5-105. This firstlayer 5-1001 may be formed from any suitable metal, such as aluminum andmay be, for example, approximately 60 nm thick. A second layer 5-1003may be formed on the first layer 5-1001. This second layer 5-1003 may beformed from any suitable metal such as titanium and may be, for example,10 nm thick. A third layer 5-1005 may be formed on the second layer5-1003. This third layer 5-1005 may be formed from any suitable ceramicsuch as titanium nitride and may be, for example, 30 nm thick. A fourthlayer 5-1007 may be formed on top of the third layer 5-1005 and alsocoating the vertical wall of the sample well. This fourth layer 5-1007may be formed from any suitable material, such as aluminum oxide and maybe approximately 5 nm thick.

The sample well of FIG. 5-16 may be formed approximately 350 nm above awaveguide 5-240 configured to carry excitation energy in the form oflight pulses to the sample well. The waveguide may be, for example, 250nm-700 nm wide. In some embodiments, the waveguide is approximately 500nm wide.

The sample well may be formed in any suitable way. For example, thefirst three layers (5-1001, 5-1003, and 5-1005) may be formed on thesubstrate 5-105 as described above. Additionally, a thin layer(approximately 5 nm) of aluminum oxide may be deposited over the firstthree layers. Then, the sample well and divot may be chemically etchedinto the layers. A second aluminum oxide layer may be deposited toconformally coat the edges of the sample well, including the bottom ofthe divot. The second aluminum oxide layer may be deposited by atomiclayer deposition, according to some embodiments. Then, the second layerof aluminum oxide may be anisotropically etched from the bottom of thedivot to expose the silicon oxide substrate.

D. Coupling Excitation Energy to Sample Well

Coupling of excitation energy to one or more sample wells of theintegrated device may occur through one or more techniques. Aspreviously discussed, in some embodiments, a waveguide is positioned tocouple with an excitation source to one or more sample wells. Asexcitation energy propagates along the waveguide, a portion of theexcitation energy may be couple to one or more sample wells through avariety of light coupling techniques. For example, the waveguide mayguide excitation energy substantially in one direction, and anevanescent wave or tail may form perpendicular to this one directionand, in some instances, be located outside the waveguide structure. Suchan evanescent tail may direct a portion of excitation energy towards oneor more sample wells. In some embodiments, the sample well layer may bedesigned and configured to direct excitation energy to a localizedregion within the sample well. The sample well may be configured toretain a sample within the localized region of the sample well such thatexcitation energy is directed towards the sample.

FIGS. 6-1A and 6-1B are cross-sectional views of an integrated deviceand provide an exemplary illustration of using a waveguide to coupleexcitation energy into a sample well. FIG. 6-1A is a cross-sectionalschematic showing a waveguide 6-104 positioned in proximity to a samplewell 6-108 in a sample well layer 6-116. Excitation energy propagatesalong the waveguide in a direction perpendicular to the field of view ofFIG. 6-1A. Proximity of a sample well to the waveguide may allowexcitation energy to couple into the sample well. FIG. 6-1B illustratesa closer view of the region of the sample well 6-108 and the sample welllayer 6-116 and shows excitation energy located within sample well6-108.

Additionally, one or more components may be formed in an integrateddevice to improve or enhance coupling of excitation energy into a samplewell. These additional components may be formed in a pixel and providecoupling of excitation energy from a waveguide into the pixel andtowards the sample well. One or more components located in a pixel mayact to tap a portion of the excitation energy from a waveguide into thepixel. Such components may include optical structures such as, gratingstructures, scattering structures, microcavities and/or nano-antennas.Features or configurations of one or more of these components may beselected for coupling a certain amount of excitation energy to eachsample well within a row or column of sample wells. A waveguideconfigured to provide excitation energy to a row of pixels may couple toa component in each pixel region to provide a portion of the excitationenergy to each pixel in the row of pixels. When a waveguide isconfigured to direct excitation energy from an excitation source towardsone or more pixels, the waveguide may be referred to as a bus waveguide.

Components positioned adjacent to a sample well may improve coupling ofexcitation energy from waveguide to sample well. Such components may becalled taps and/or microcavities. A microcavity may deflect a portion ofthe excitation energy from the waveguide such that excitation energyreaches the sample well. One or more microcavities may be used to coupleexcitation energy to a sample well. The one or more microcavities mayreduce loss of excitation energy from the waveguide, including metallicloss. One or more microcavities may act as a lens to focus excitationenergy to the sample well. In some embodiments, one or moremicrocavities may improve directing luminescence from a marker in thesample well towards the sensor. The microcavities may have acylindrical, convex, concave, rectangular, spherical, or ellipticalconfiguration or any other suitable shape. A microcavity may be formedfrom any suitable material. In some embodiments, a microcavity mayinclude silicon nitride.

One or more microcavities may overlap with at least a portion of awaveguide to direct excitation energy towards the sample well whenviewed from the top of the integrated device where the sample wells arepresent. The thickness of a waveguide may be configured to reduce lossof excitation energy and improve coupling of excitation energy to one ormore microcavities. In some embodiments, microcavities along a row ofsample wells may vary in strength of coupling between the waveguide toeach sample well. The microcavities may increase coupling along thepropagation direction of the excitation energy in order to accommodate areduced power in the waveguide as excitation energy is directed out ofthe waveguide to each sample well. In some embodiments, one or moremicrocavities are adjacent to the sample well. There may be an offsetdistance between the location of the center of a sample well and thecenter of a microcavity. In other embodiments, one microcavity islocated below a sample well such that at least a portion of the samplewell and a portion of the microcavity overlap when viewed from the topof the integrated device where the sample wells are present.

FIG. 6-2A illustrates a planar view of an exemplary pixel region havingwaveguide 6-204 positioned proximate to sample well 6-208 such thatwaveguide 6-204 and sample well 6-208 are non-overlapping. As shown inFIG. 6-2A, microcavity 6-218 b has a smaller cross-sectional diameterthan microcavity 6-218 a. Microcavities 6-218 a and 6-218 b arepositioned relative to waveguide 6-204 and sample well 6-208 to coupleexcitation energy from waveguide 6-204 to sample well 6-208. Togethermicrocavities 6-218 a and 6-218 b deflect a portion of excitation energywithin the waveguide to the sample well. A portion of microcavity 6-218b is positioned to overlap with waveguide 6-204. Microcavity 6-218 b ispositioned proximate to microcavity 6-218 b to provide sufficientcoupling between the two microcavities. Microcavity 6-218 a ispositioned closer to sample well 6-208 than microcavity 6-218 b. In sucha configuration microcavities 6-218 a and 6-218 b may act as a tap tocouple a portion of excitation energy from waveguide 6-204 to samplewell 6-208. FIG. 6-2B illustrates an elevation view from the perspectivealong line A-A′ shown in FIG. 6-2A. Microcavities 6-218 a and 6-218 bare positioned adjacent to layer 6-216 forming sample well 6-208. Layer6-216 may include a metal (e.g., aluminum). In this exemplaryembodiment, microcavities 6-218 a and 6-218 b have cylindrical shapeswhere an end of the cylinder is positioned at or at least proximate to asurface of layer 6-218. A distance between waveguide 6-204 and an edgeof microcavity 6-218 a and/or microcavity 6-218 b may allow for adesired amount of excitation energy to couple into sample well 6-218.FIG. 6-2C illustrates an elevation view from the perspective along lineB-B′ shown in FIG. 6-2A. Microcavity 6-218 b is positioned to overlapwith waveguide 6-204, while microcavity 6-218 a is positioned to notoverlap with waveguide 6-204.

FIG. 6-3A-D illustrate planar views of further exemplary configurationsof one or more microcavities with respect to a sample well and waveguidein an integrated device. The one or more microcavities may overlap witha portion of the waveguide. In some instances, a microcavity is locatedbelow the sample well. FIG. 6-3A shows microcavity 6-306 overlappingwith sample well 6-308 with respect to waveguide 6-304. FIG. 6-3B showscavity 6-316 b overlapping with sample well 6-318 with respect towaveguide 6-314. In other embodiments, a microcavity is located at adistance offset from the sample well such that the microcavity andsample well do not overlap. FIG. 6-3B shows microcavity 6-318 apositioned from sample well 6-318 such that there is no overlappingregion between microcavity 6-316 a and sample well 6-318. Similarly,FIG. 6-3C shows microcavity 6-326 positioned from sample well 6-328 withrespect to waveguide 6-324 such that there is no overlapping regionbetween microcavity 6-316 a and sample well 6-318. In some embodiments,multiple microcavities may be positioned offset from a sample well. FIG.6-3D shows microcavities 6-336 a and 6-336 b positioned from sample well6-338 with respect to waveguide 6-334 such that there is no overlappingregion between sample well 6-338 and either 6-336 a or 6-336 b. Themicrocavity design, position with respect to the sample well, and/orposition with respect the waveguide may be determined based on reducingthe overall coupling loss and improving coupling efficiency between thewaveguide and the sample well.

FIG. 6-4 shows a cross-sectional view of microcavity 6-418 positioned tocouple excitation energy from waveguide 6-404 to sample well 6-408 inlayer 6-416. One or more dimensions of microcavity 6-418 and/orwaveguide 6-404 may provide a desired amount of coupling. A waveguide ofan integrated device, such as waveguide 6-404, has cross-sectionalheight, t, which may be approximately 50 nm, approximately 100 nm,approximately 150 nm, approximately 160 nm, approximately 170 nm, orapproximately 200 nm. Microcavity 6-418 has a cross-sectional dimensionD, such as diameter if microcavity has a cylindrical shape.Cross-sectional dimension, D, may be approximately 550 nm, approximately600 nm, approximately 650 nm, approximately 700 nm, approximately 750nm, or approximately 800 nm. Microcavity 6-418 has cross-sectionalheight, h, which may be approximately 450 nm, approximately 500 nm,approximately 550 nm, or approximately 600 nm. The position ofmicrocavity 6-418 relative to waveguide 6-408 and sample well 6-408 mayallow for coupling of excitation energy from waveguide 6-408 to samplewell 6-408. A distance between microcavity 6-418 and waveguide 6-408that is perpendicular to propagation of light in waveguide 6-408, shownas x in FIG. 6-4, may be approximately 300 nm, approximately 350 nm,approximately 400 nm, approximately 450 nm, or approximately 500 nm. Adistance of microcavity 6-418 offset from sample well 6-408, shown as din FIG. 6-4, may be approximately 500 nm, approximately 550 nm,approximately 600 nm, approximately 650 nm, or approximately 700 nm.Layer 6-416 including sample well 6-408 may have a cross-sectionalheight, y, of approximately 50 nm, approximately 100 nm, orapproximately 150 nm. Sample well 6-408 may have a cross-sectionaldimension, such as a diameter, of approximately 75 nm, approximately 100nm, approximately 125 nm, approximately 150 nm, or approximately 175 nm.

In an exemplary embodiment, waveguide 6-404 has a cross-sectional widthof 667 nm and a cross-sectional height of 100 nm. Microcavity 6-418 hasa dimension h of 591 nm, a cross-sectional diameter of 750 nm, and ispositioned such that distance x from waveguide 6-404 is 398 nm anddistance d from sample well 6-408 is 693 nm. Such a pixel configurationmay have a total coupling loss of 0.28%, which if implemented in anintegrated device having 256 pixels, may have a transmission loss ofapproximately 51% and a metallic loss of approximately 11%.

In another exemplary embodiment, waveguide 6-404 has a cross-sectionalwidth of 639 nm and a cross-sectional height of 100 nm. Microcavity6-418 has an ellipsoid shape with one cross-sectional dimension of 600nm, another cross-sectional dimension of 639 nm, a dimension h of 512nm. Such a pixel configuration may have a total coupling loss ofapproximately 0.77%, which if implemented in an integrated device having64 pixels, may have a transmission loss of approximately 30% and a metalloss of approximately 35%.

FIG. 6-5 illustrates simulation results of propagation of light in aconfiguration having two microcavities 6-536 a and 6-536 b relative tosample well 6-538 and waveguide 6-534. As shown in FIG. 6-5, the darkregions correspond to higher intensity of light which extend fromwaveguide 6-534 to sample well 6-538 and supported by microcavities6-536 a and 6-536 b.

In another exemplary embodiment, waveguide has a cross-sectional widthof 700 nm and a cross-sectional height of 100 nm. A microcavity iscylindrical with a diameter of 600 nm and has dimension h of 650 nm. Themicrocavity is positioned in proximity to the waveguide and the samplewell such that there is a coupling of 0.148%, a metallic loss of0.09856%, a return loss of 0.1225%, and a radiation loss of 0.1270%.FIGS. 6-6A to 6-6C illustrate simulations of light propagation throughsuch an embodiment. FIG. 6-6A illustrates a cross-sectional view ofsample well 6-638, microcavity 6-636, and waveguide 6-634. Waveguide6-634 supports a mode of light that propagates along waveguide 6-634,and microcavity 6-636 is positioned to act as a tap to couple some ofthe excitation energy into sample well 6-638. In FIG. 6-6A, darkerregions indicates areas having higher light intensity. FIG. 6-6Billustrates a planar view of microcavity 6-636 and sample well 6-638,where light is directed by microcavity 6-636 towards sample well 6-638.FIG. 6-6C illustrates a transverse cross-sectional view showing lightfrom wavegide 6-634 coupling to sample well 6-638.

FIG. 6-6D illustrates a planar view of simulations for another exemplaryconfiguration having waveguide 6-654, microcavity 6-656, and sample well6-658. In this configuration, sample well 6-658 overlaps microcavity6-656, and microcavity 6-658 and waveguide 6-654 overlap. FIG. 6-6Dshows of light, where dark regions indicate higher intensity,propagation to sample well 6-658 supported by microcavity 6-656.

Some embodiments relate to a microcavity positioned between a samplewell layer and a waveguide such that the microcavity is offset from asurface of the sample well layer. The microcavity may be offset from asurface of the sample well layer in a direction perpendicular to thepropagation of light along the waveguide. In some embodiments, themicrocavity may also be offset from a location of a sample well suchthat the sample well and microcavity are non-overlapping. Themicrocavity may be sized and shaped to provide a desired amount ofcoupling of excitation energy from the waveguide to the sample well. Insome embodiments, the microcavity may have a longer dimension along adirection parallel to a direction of light propagation through thewaveguide than in a dimension perpendicular to light propagation throughthe waveguide.

FIG. 6-7A shows a cross-sectional view of an integrated device havingmicrocavity 6-718 positioned proximate to sample well 6-708 andwaveguide 6-704. Sample well 6-708 is formed in sample well layer 6-716.Microcavity has dimension D, which is parallel to the direction of lightpropagating through waveguide 6-704 and dimension h, which isperpendicular to the direction of light propagating through waveguide6-704. In some embodiments, dimension D of microcavity 6-718 is largerthan dimension h of microcavity 6-718. Microcavity 6-718 may have adimension h of approximately 100 nm, approximately 150 nm, orapproximately 200 nm. Microcavity 6-718 may have a dimension D ofapproximately 500 nm, approximately, 750 nm, or approximately 1000 nm.Microcavity 6-718 is positioned a distance along the y-direction from asurface of waveguide 6-704 having dimension x1 and a distance along they-direction from a surface of sample well layer 6-716 having dimensionx2. In some embodiments, dimension x2 is smaller than dimension x1.Microcavity 6-718 may be offset from sample well layer 6-716 such thatx2 is approximately 200 nm, approximately 300 nm, or approximately 400nm, in some embodiments.

In some embodiments, microcavity 6-718 is offset from sample well 6-708by a distance along a direction parallel to a direction of lightpropagation through waveguide 6-704. Microcavity 6-718 may be offsetfrom sample well 6-708 by a center-to center distance, d. In someembodiments, microcavity 6-718 is offset from sample well 6-708 suchthat microcavity 6-718 and sample well 6-708 are non-overlapping. Insome embodiments, microcavity 6-718 may be positioned such that an edgeof microcavity 6-718 proximate to sample well 6-708 is offset fromsample well 6-708 by a distance. An offset distance between an edge ofmicrocavity 6-718 and sample well 6-708 may be approximately 50 nm,approximately 100 nm, approximately 150 nm, or approximately 200 nm.

FIG. 6-7B shows a cross-sectional view of intensity of light asexcitation energy propagates through waveguide 6-704 and couples tosample well 6-708 in sample well layer 6-716 via microcavity 6-718,similar the configuration shown in FIG. 6-7A. Excitation energy travelsalong the z-direction through waveguide 6-704. As shown in FIG. 6-7B, aportion of excitation energy (shown as dark lines) reaches sample well6-708 by coupling to microcavity 6-718. Since excitation energycontinues to propagate along waveguide 6-704, microcavity 6-718 acts asa tap by directing a portion of the excitation energy away fromwaveguide 6-704 and towards sample well 6-708.

FIG. 6-7C shows a planar view of microcavity 6-718 positioned proximateto sample well 6-708. Excitation energy (shown as dark regions)propagates through microcavity and is directed to within sample well6-708. As shown in FIG. 6-7C, microcavity 6-718 may have a rectangularshape with curved edges that act to direct light towards sample well6-708. An edge of microcavity 6-718 may have a radius of curvature toallow a desired level of coupling of excitation energy to sample well6-708. A first edge of microcavity 6-718 proximate to sample well 6-708may have a smaller radius of curvature than a second edge of microcavity6-718 opposite the first edge.

Some embodiments relate to an microcavity positioned between a samplewell and a waveguide such that the microcavity overlaps with the samplewell and is a distance offset from the sample well. The microcavity andsample well may overlap in a direction perpendicular to a direction oflight propagation along the waveguide. The microcavity may act toenhance coupling of excitation energy into the sample well. In someembodiments, the microcavity may be aligned to the sample well with asubstantial center-to-center alignment between the microcavity andsample well. In some embodiments, the microcavity may be positionedcloser to the waveguide than to the sample well. In some embodiments,the microcavity may be positioned on a surface of the waveguide.

FIG. 6-7D shows a cross-sectional view of an integrated device havingsample well 6-728, waveguide 6-734, and microcavity 6-738 positionedbetween sample well 6-728 and waveguide 6-734. Sample well 6-728 isformed in sample well layer 6-738 and may extend beyond sample welllayer 6-738 to include a divot in layer 6-732 of the integrated device.Layer 6-732 may have dimension h along the x-direction as shown in FIG.6-7D. Microcavity 6-738 may be positioned within layer 6-732 such thatthere is a portion of layer 6-732 between microcavity 6-738 and samplewell 6-728 and a portion of layer 6-732 between microcavity 6-738 andwaveguide 6-734. As shown in FIG. 6-7D, microcavity 6-738 is positioneda distance along the y-direction from sample well 6-728 having dimensiond1. Microcavity 6-738 is positioned a distance along the y-directionfrom waveguide 6-734 having dimension d2. In some embodiments, dimensiond2 is smaller than d1. In some embodiments, microcavity 6-738 ispositioned at a surface of sample well 6-728 and within layer 6-732 suchthat dimension d1 is equal to zero.

One or more dimensions of a waveguide of an integrated device may varyalong the length of the waveguide in the direction of light propagationthrough the waveguide. Varying one or more dimensions along thewaveguide may improve coupling efficiency and substantial uniformity inthe amount of excitation energy provided by the waveguide to a pluralityof sample wells. In some embodiments, the cross-sectional width of awaveguide may vary along a row or column of pixels. The waveguide mayinclude a taper such that the cross-sectional width of the waveguidedecreases along the direction of propagation of excitation energythrough the waveguide. FIG. 6-8A shows a planar view of waveguide 6-804in an integrated device. Excitation source 6-802 couples to waveguide6-804 using one or more techniques described herein (e.g., gratingcouplers, star couplers, MMI splitters). Waveguide 6-804 is tapered suchthat a dimension of waveguide 6-804 along the x-direction shown in FIG.6-8A decreases along a dimension of waveguide 6-804 along thez-direction or a direction light propagation through waveguide 6-804. Inthis manner, waveguide 6-804 has a larger cross-sectional width (alongthe x-direction) proximate to incident excitation source 6-802 than at alocation further along the length (along the z-direction) of waveguide6-804. Positioning waveguide 6-804 to couple with sample wells in a rowof pixels may provide a configuration sufficient for coupling a desiredamount of excitation energy into each sample well. Since a portion ofexcitation energy is coupled out of waveguide 6-804 for each samplewell, the amount of excitation energy propagated by waveguide 6-804reduces along the z-direction. Along waveguide 6-804, the amount ofpower reduces as excitation energy is coupled out to the sample wells.By reducing the cross-sectional width of waveguide 6-804 may propagateexcitation energy further along waveguide 6-804 than in a waveguidewithout such a taper.

FIG. 6-8B shows a cross-sectional view of waveguide 6-804 shown in FIG.8A. A dimension of waveguide 6-804 along the y-direction remainssubstantially similar such that a distance along the y-direction betweensample wells 6-808 and waveguide 6-804 is substantially constant alongthe length of waveguide 6-804. Dotted curved lines illustrate the spreadof excitation energy as it propagates along waveguide 6-804 in thez-direction. As the cross-sectional width of waveguide 6-808 narrows,the spread of excitation energy becomes broader and may compensate forreduced power. In this manner, the cross-sectional width of waveguide6-802 may balance reduction in power of excitation energy propagated bywaveguide 6-802 such that a sufficient amount of excitation energy isdelivered to each sample well in a row of pixels for a desiredperformance of an integrated device.

In some embodiments, a tapered waveguide may be configured for a similarcoupling efficiency for a row of pixels where pixels in the row includea microcavity and a sample well. A combination of varying one or moredimensions of the tapered waveguide and/or the microcavity mayaccommodate reduction in power of excitation energy along the length ofa waveguide as excitation energy is coupled to each sample well in therow.

In some embodiments, one or more sensors may be positioned relative to awaveguide to measure excitation energy propagating along the waveguide.Additional structures (e.g., gratings) may be positioned to tap out atleast a portion of excitation energy to a sensor. As shown in FIG. 6-8B,monitoring sensors 6-812 a and 6-812 b are positioned proximate to theends of waveguide. Gratings 6-810 a and 6-810 b are positioned on a sideof waveguide 6-808 opposite to sensors 6-812 a and 6-812 b. Gratings6-810 a and 6-810 b may be configured to direct excitation energy inwaveguide 6-808 towards sensors 6-812 a and 6-812 b, respectively. Thecombination of grating 6-810 a and sensor 6-812 a may monitor excitationenergy input into waveguide 6-804. The combination of grating 6-810 band sensor 6-812 b may monitor excitation energy, if any, excitationenergy at an end of waveguide 6-804 that remains after couplingexcitation energy to a row of sample wells 6-808. In this manner,sensors 6-812 a and 6-812 b may monitor power in waveguide 6-804 at aninput end, and output end, and/or any suitable location along waveguide6-808. Sensors 6-812 a and 6-812 b may detect information including alevel of power at a location along waveguide 6-808. This information maybe used to control aspects of components that act to align an excitationsource to the integrated device and/or a power of the excitation source.

In some embodiments, a waveguide may couple to a sample well by anevanescent field. In some embodiments, a bullseye grating structurehaving concentric grating rings positioned proximate to a sample wellmay improve coupling of excitation energy from the waveguide to thesample well. In some embodiments, the waveguide may include a regionhaving a reduced cross-sectional width in the vicinity of a sample wellsuch that an evanescent field from the excitation energy propagating inthe waveguide couples to the sample well. For a row of pixels, awaveguide may include multiple regions having a reduced cross-sectionalwidth along the length of the waveguide to improve coupling uniformityand efficiency among sample wells in the row. In this manner, thewaveguide may be considered to have pinched sections at certainlocations along the waveguide.

The layer of an integrated device having one or more sample wells mayinterfere with propagation of light through a waveguide. In someembodiments, the sample well layer is formed of a metal (e.g.,aluminum). It may be desirable to position the sample well layer at acertain distance from the waveguide to reduce loss and improveperformance of the device. These techniques may allow for a desiredperformance achieved by positioning the sample well layer at a certaindistance from the waveguide while allowing a sample well in the layer toreceive a sufficient amount of excitation energy.

FIG. 6-9A shows a planar view of waveguide 6-904 relative to a bullseyegrating structure 6-910. Bullseye grating structure 6-910 comprises aplurality of concentric circular gratings centered at a sample well.Waveguide 6-904 includes a pinched region where the cross-sectionalwidth of waveguide 6-904 is smaller at a location proximate to thecenter of bullseye grating structure 6-910. The pinched region ofwaveguide 6-904 may be formed by tapering a certain distance alongwaveguide 6-904. The length of waveguide 6-904 that is tapered may be asuitable amount to reduce excitation energy loss and/or improve couplingefficiency. A waveguide may include multiple pinched regions, where eachpinched region is associated with a sample well. The multiple pinchedregions may have one or more dimensions that vary along the length ofthe waveguide to provide suitably uniform power and coupling efficiencyto each of the sample wells. FIG. 6-9B shows a cross-sectional view ofwaveguide 6-904 configured to couple excitation energy to sample well6-908. Bullseye grating 6-910 is substantially centered at sample well6-908 and configured to couple excitation energy from waveguide 6-904 tosample well 6-908. The cross-sectional width of waveguide 6-904 has apinched region proximate to sample well 6-908 such that the spread ofthe field of excitation energy broadens as the cross-sectional widthnarrows. The dotted line indicates the extent to which the field ofexcitation energy extends perpendicularly from waveguide 6-904 in they-direction. At a location along waveguide 6-904 that overlaps withsample well 6-908, the field of excitation energy is at a certainlocation to allow coupling of the excitation energy to sample well6-908.

One or more dimensions of the taper in a pinched region and/or a shapeof the taper in the pinched region of a waveguide may provide a desiredlevel of coupling efficiency. FIG. 6-10 illustrates a plot of total lossfor different tapering profiles as a function of length over which thetaper profile occurs or taper length to indicate the amount of losscaused by the tapering process. As shown by FIG. 10, having a moregradual tapering of the pinched region may improve coupling efficiencyby reducing total coupling loss. Tapering the cross-sectional width from0.5 microns to 0.3 microns for either linear or s-shaped configurationshas lower total loss than if the cross-sectional width is tapered from0.5 microns to 0.2 microns. Additionally, increasing the length overwhich the cross-sectional width is reduced may improve total couplingloss. In some embodiments, a waveguide may include a pinched regionwhere a cross-sectional width of the waveguide varies from approximately0.3 microns to approximately 0.5 microns over a length along thewaveguide of approximately 20 microns, approximately 40 microns,approximately 50 microns, approximately 60 microns, approximately 80microns, or approximately 100 microns.

In some embodiments, a waveguide may couple to a sample well bydecreasing a distance between a sample well and the waveguide within aregion of the sample well. Such a configuration may allow for portionsof the waveguide to be positioned a certain distance from a sample welllayer to provide a desired efficiency and loss of excitation energy.FIG. 6-11A shows a cross-sectional view of waveguide 6-1104 positionedwith respect to layer 6-1102 having sample well 6-1108. Bullseye gratingis formed adjacent to layer 6-1102 and substantially centered at samplewell 6-1108. In such embodiments, the waveguide has a substantiallyuniform cross-sectional thickness and the field of excitation energyextending from waveguide 6-1104 is approximately uniform along thelength of waveguide 6-1104. By forming layer 6-1102 such that samplewell 6-1108 and waveguide 6-1104 have a certain distance along they-direction, the excitation energy may couple to sample well 6-1108.Bullseye grating 6-1110 may act to further couple excitation energy fromwaveguide 6-1104 to sample well 6-1108. Other regions along waveguide6-1104 separate from sample well 6-1108 may have layer 6-1102 at largerdistance along the y-direction than in a vicinity proximate to samplewell 6-1108. Such a configuration may be considered a sample well dipconfiguration and, in some embodiments, provide a coupling efficiency ofapproximately 0.3%.

Some embodiments related to forming one or more layers of material on awaveguide to improve coupling of excitation energy from the waveguide toa sample well. FIG. 6-11B shows a cross-sectional view of sample well6-1112 formed from sample well layer 6-1116. As shown in FIG. 6-11B,sample well layer 6-1116 includes a region having an opening of samplewell layer 6-1116 which forms sample well 6-1112. The region of samplewell layer 6-1116 is offset by dimension h from a surface of theintegrated device. Dimension h may be greater than approximately 200 nm,approximately 250 nm, approximately 300 nm, approximately 350 nm, orapproximately 400 nm. The offset region of sample well layer 6-1116 maybe formed by etching a portion of layer 6-1120 and forming sample welllayer 6-116 over layer 6-1120. The region of sample well layer 6-1116that includes sample well 6-1112 is positioned a distance d1 fromwaveguide 6-1118 along the y-direction. Distance d1 may be approximately250 nm, approximately 300 nm, approximately 350 nm, approximately 400nm, or approximately 450 nm. Layer 6-1114 is formed adjacent towaveguide 6-1118. Layer 1114 may have a higher refractive index thatlayer 6-1120 and a lower refractive index than waveguide 6-1118. In someembodiments, layer 6-1114 includes a plurality of layers havingdifferent materials. In an exemplary embodiment, waveguide 6-1118includes silicon nitride and layer 6-1114 includes aluminum oxide and/ortitanium oxide. Layer 6-1114 is positioned a distance d2 from samplewell layer 6-1116. Distance d2 may be approximately 50 nm, approximately100 nm, approximately 150 nm, approximately 200 nm, or approximately 250nm. FIG. 6-11C shows a cross-sectional view along line A-A′ shown inFIG. 6-11B. As shown in FIG. 6-11C, waveguide 6-1118 may have a lateraltransverse dimension along the x-direction that overlaps with the offsetregion of sample well layer 6-1116 that includes sample well 6-1112.Layer 6-1114 may be formed on waveguide 6-1118 such that layer 6-1114contacts a plurality of sides of waveguide 6-111C.

Additionally or alternatively, the spacing between pixels in a row alonga waveguide may be selected to reduce waveguide loss. Each pixel mayinclude a sample well, one or more sensors, and/or a bullseye grating.The spacing between rows of pixels may be selected to accommodate otherintegrated device components, such as circuitry. FIG. 6-12 shows aplanar view of an integrated device having pixels 6-1208 arranged in arectangular array with waveguides 6-1204 configured to deliverexcitation energy to a row of pixels 6-1208. FIG. 6-12 shows aconfiguration of the pixel array where spacing between pixels in a row,x, is smaller than spacing between rows, y. By positioning pixels in arow at a certain distance, waveguide loss along the waveguide may bereduced. Having a larger spacing between rows may reduce interference ofone row of pixels on another row and/or provide suitable spacing toaccommodate additional components on the device, including circuitrycomponents.

The whole pixel array of an integrated device may have any suitablenumber of pixels and any suitable arrangement of pixels. In someembodiments, a pixel array may have a square and/or rectangularconfiguration. In some embodiments, the array of pixels may berectangular and have a length parallel to the waveguides that is longerthan the length perpendicular to the waveguides. In other embodiments,there may be more waveguides and/or rows of pixels, and the length ofeach row of pixels parallel to the waveguides may be shorter than thelength perpendicular to the waveguide.

Some embodiments relate to an integrated device with a waveguide havinga variable dimension in a direction perpendicular to light propagationthrough the waveguide and perpendicular to a surface of the integrateddevice with one or more sample wells. In some embodiments, the dimensionof the waveguide may vary to be larger in a region proximate to a samplewell but non-overlapping with the sample well. In some embodiments, thedimension of the waveguide may decrease in a region of the waveguidethat overlaps with the sample well. An integrated device may include awaveguide configured to couple with a row of sample wells, where thevariation in the dimension of the waveguide allows for a substantiallysimilar amount of excitation energy to couple to each sample well.

FIG. 6-13 shows a cross-sectional view of an integrated device havingsample well 6-1308 formed in sample well layer 6-1316. Waveguide 6-1304includes region 6-1310 having a dimension x1 along the y-direction x1that is larger than a portion of waveguide 6-1304 that overlaps withsample well 6-1308 having dimension x2 along the y-direction. As shownin FIG. 6-13, region 6-1310 is offset from sample well 6-1308 and form aridge in waveguide 6-1304. Region 6-1310 may be sized and shaped toprovide a desired amount of excitation energy from waveguide 6-1304 tosample well 6-1308. FIG. 6-14 shows a cross-sectional view of anintegrated device having sample well 6-1408 formed in sample well layer6-1416. Waveguide 6-1404 includes region 6-1410 that overlaps withsample well 6-1408. Waveguide in region 6-1410 has a dimension x2 alongthe y-direction that is smaller than portions of the waveguide outsideof region 6-1410, which have a dimension x1 along the y-direction. Inthis configuration waveguide 6-1404 includes a dimension smaller withinregion 6-1410 that overlaps with sample well 6-1408.

Although FIGS. 6-13 and 6-14 show a single sample well, waveguides6-1304 and 6-1404 may be configured to couple to a row of sample wellsand have a region similar to regions 6-1310 and 6-1404 positionedproximate to each sample well. In some embodiments, the regionspositioned proximate to the plurality of sample wells in the row mayvary in size and/or shape such that a substantially similar amount ofexcitation energy couples to each sample well. In this manner, regionsof a waveguide may be configured to accommodate for decreased excitationenergy propagating along the waveguide along a row of sample wells.

E. Directing Emission Energy to Sensor

An integrated device may include one or more components positionedbetween a sample well and a sensor of a pixel to improve collection ofluminescence by the sensor from a sample in the sample well. Suchcomponents may improve the signal-to-noise ratio of the luminescencesignal to a background signal to provide improved detection of aluminescent marker. Some components may direct luminescence from asample well to a corresponding sensor in a pixel. In some embodiments, acomponent may provide both suitable coupling of excitation energy to asample well and coupling of luminescence out of the sample well. Othercomponents (e.g., filters) may reduce excitation energy and other lightnot associated with the sample and/or marker from contributing to asignal acquired by the sensor.

A bullseye grating may be formed from concentric grating rings around asample well. A bullseye grating may couple with a sample well to improvepropagation of luminescence out of the sample well. The bullseye gratingstructure may direct luminescence towards a sensor in a pixel having thesample well. In some embodiments, an effective diameter of theluminescence directed by a bullseye grating is approximately 5 microns.

FIG. 7-1A shows a planar view of bullseye 7-110 configured to directluminescence from sample well 7-108. Bullseye structure 7-110 includes aplurality of concentric gratings. The concentric gratings may alignsubstantially with the center of sample well 7-108. Waveguide 7-104 maybe positioned of overlap with at least a portion of bullseye structure7-110 and sample well 7-108. Waveguide 7-104 includes a tapered region,which may allow for luminescence to pass to a sensor positioned withinthe pixel having sample well 7-108 and a desired level of collection ofluminescence by the sensor. The tapered region may allow for reducedinterference of waveguide 7-104 on the collection of luminescence by thesensor. In some embodiments, a bullseye grating may be configured todirect more than one characteristic luminescence energies or wavelengthstowards a sensor. FIG. 7-1B shows a cross-sectional view of bullseyegrating configured to direct luminescence from sample well 7-108. Thedotted lines indicate the spread of two characteristic wavelengths, λ1and λ2, that may be coupled out of sample well 7-108. The twocharacteristic wavelengths, λ1 and λ2, may be characteristic wavelengthsemitted from different markers used to label a sample. FIG. 7-1C shows across-sectional view of bullseye grating 7-110 at a transversecross-sectional view of waveguide 7-104. Bullseye grating 7-110 andwaveguide 7-104 are configured such that luminesce emitted from samplewell 7-108 is spread to allow luminescence energy to pass through aregion of the integrated device other than waveguide 7-104. Bullseyegrating 7-110 may direct luminescence over a certain distance fromsample well 7-108 that is greater than if the bullseye grating was notpresent. In some embodiments, bullseye grating 7-110 may reduce overallscattering of luminescence. Bullseye grating 7-110 may be configuredboth for coupling excitation energy to sample well 7-108 and to directluminescence energy out of sample well 7-108.

Microcavities provided for coupling of a waveguide and a sample well toallow propagation of excitation energy to the sample well may alsodirect luminescence from the sample well to a sensor. FIG. 7-2A shows across-sectional view of waveguide 7-204 offset from sample well 7-208.Microcavity 7-211 is configured to direct luminescence from sample well7-208. FIG. 7-2A shows intensity of luminescence emitted by sample well7-208 where regions having a higher intensity of luminescence aredarker. Microcavity 7-211 may direct luminescence at an angle away fromwaveguide 7-204 to reduce scattering of the luminescence by waveguide7-204. FIG. 7-2B illustrates a plot luminescence emitted from a samplewell as a function of angle. As shown in FIG. 7-2B, microcavity 7-211may direct luminescence at an angle of approximately 15 degrees from they-direction. One or more microcavities may direct luminescence at anangle of approximately 5 degrees, approximately 10 degrees,approximately 15 degrees, approximately 20 degrees, approximately 25degrees from the y-direction. In some embodiments, a sensor may beoffset from the sample well in order to receive the luminescencedirected at such an angle. Microcavity 7-211 may also be configured tocouple excitation energy from waveguide to the sample well such thatmicrocavity 7-211 provides both coupling of excitation energy to asample well and directs luminescence out of the sample well. In someembodiments, more than one microcavity may be formed to coupleluminescence out of a sample well.

A baffle having an opening centered on a sensor may be formed between asample well and the sensor. As shown in FIG. 4-2, baffle layer 4-226 ispositioned between sample well 4-22 and sensor layer 4-230. A baffle ina pixel may be designed to suppress collection of energy besidesluminescence for the pixel. A baffle associated with a sample well and asensor may allow for luminescence from the sample well to reach thesensor while reducing luminescence from neighboring pixels, excitationenergy, and other energy not associated with the luminescence from thesample well associated with the sensor. A dimension of the opening ofthe baffle may be configured to allow luminescence directed by abullseye on the same pixel. As shown in FIG. 4-2, baffle 4-226 hasopenings along the z-direction to allow luminescence to pass through tosensors positioned in layer 4-230. The material of a baffle may beselected for certain optical properties, such as reducing transmissionof certain light wavelengths or energies at certain incident angles. Insome embodiments, a baffle may be formed by multiple layers of materialshaving different refractive indices. A baffle may include one or morelayers of silicon, silicon nitride (Si₃N₄), silicon, titanium nitride(TiN), and aluminum (Al). A layer configured to form a baffle may have across-sectional height of approximately 20 nm, approximately 20 nm,approximately 50 nm, approximately 60 nm, approximately 70 nm,approximately 80 nm, or approximately 90 nm.

In an exemplary embodiment, a baffle includes four layers: a layer ofsilicon nitrate having a cross-sectional thickness of 22.34 nm, a layerof amorphous silicon having a cross-sectional thickness of 81.34 nm, alayer of titanium nitride having a cross-sectional thickness of 30 nm,and a layer of aluminum having a cross-sectional thickness of 50 nm.Absorption and reflectance of an exemplary baffle design is illustratedin FIG. 7-3, which shows absorbance and reflectance at differentincident angles of 0, 20, and 35 degrees for both p and s polarizations.As shown in FIG. 7-3, the baffle has varying levels of absorptance fordifferent incident angles to the baffle and has a larger absorptance atlarger incidence angles.

Filtering components may be formed between a waveguide and a sensor forreducing collection of excitation energy by the sensor. Any suitablemanner for filtering excitation energy may be provided. Techniques forfiltering the excitation energy may include filtering based on one ormore characteristics of light. Such characteristics may includewavelength and/or polarization. Filtering components may selectivelysuppress scattered excitation energy while allowing luminescence to passthrough to the sensor. Layer 4-228 shown in FIG. 4-2 may include one ormore filtering components described herein.

A polarization filter configured to reflect and/or absorb light of aparticular polarization may act as a filter for in an integrated device.A polarization filter may be used in systems where excitation energypropagating through a waveguide has a certain polarization andluminescence emitted by a marker may have a different polarization thanthe excitation energy. The polarization excitation filter may beconfigured to filter the polarization of the excitation energy andthereby reduce excitation energy from being collected by a sensor. Insome embodiments, a polarization filter may be configured to absorbTE-polarized light when the excitation energy provided by the excitationis TE polarized. The polarization filter may consist of a patternedwire-grid polarizer. In some embodiments, the wires in the wire-grid arealuminum. The orientation of the wires may be aligned parallel along adirection perpendicular to the propagation direction of one or morewaveguides. When the excitation energy is TE-polarized, the orientationof the wires may be aligned with the oscillating electric field of theTE-polarized excitation energy. Fabrication of such a wire-gridpolarizer may provide a suitable filter while negligibly contribute tothe thickness of the overall integrated device. Such a wire-gridpolarizer may have a loss of scattered excitation energy ofapproximately greater than 10 dB and a loss of luminescence ofapproximately 3 dB. In some embodiments, openings in the wire-gridfilter where the wires are not present may align with one or moreunderlying sensors. The openings may have a dimension that is smaller orlarger than a dimension of one or more sensors. An opening may allow forluminescence from a sample well of a pixel to be collected by a sensorin the pixel while the surrounding wire-grid pattern may providefiltering of excitation energy.

FIG. 7-4A shows a planar view of a polarization excitation filter havinga grid comprising a plurality of wires 7-406. The wire grid has openings7-408 that form pixel regions in a pixel array of an integrated device.Waveguide 7-404 may be positioned to pass through a row of openings7-408. FIG. 7-4B shows a cross-sectional view along line A-A′ as shownin FIG. 7-4A. Wires 7-406 are positioned between waveguide 7-404 andsensor 7-410. Opening 7-408 in the wires 7-406 is positioned to at leastpartially overlap with sensor 7-410. In this manner, opening 7-408 andsensor 7-410 may be included in the same pixel of an integrated device.Opening 7-408 may allow luminescence energy to pass through such thatsensor 7-410 may detect the luminesce, while reducing the amount ofexcitation energy detected by sensor 7-410.

An integrated device may include a wavelength filter configured toreflect light of one or more characteristic wavelengths and allowtransmission of light having a different characteristic wavelength. Insome embodiments, light reflected by a wavelength filter may have ashorter characteristic wavelength than the light transmitted by thewavelength filter. Such a wavelength filter may reflect excitationenergy and allow transmission of luminesce since excitation energy usedto excite a marker typically has a shorter characteristic wavelengththan luminesce emitted by the marker in response reaching an excitationstate by the excitation energy.

A wavelength filter may include one or more layers having one or morematerials. A wavelength filter may include titanium dioxide (TiO₂)and/or silicon dioxide (SiO₂). In some embodiments, a wavelength filtermay include a stack of multiple layers having alternating layers of ahigh index of refraction material and a low index of refractionmaterial. The cross-sectional thickness of each layer may be anysuitable thickness and, in some instances, may be configured to reflectand/or transmit a particular wavelength of light. The cross-sectionalthickness of the layers may be approximately a quarter of the wavelengthof the light transmitted by the wavelength filter. A thickness of alayer of a wavelength filter may be approximately 10 nm, approximately40 nm, approximately 50 nm, approximately 60 nm, approximately 70 nm,approximately 80 nm, approximately 90 nm, approximately 100 nm,approximately 110 nm, approximately 130 nm, or approximately 150 nm. Insome embodiments, a layer of an wavelength filter may have less than 1%variation in thickness across the layer, providing suitable thicknessuniformity within the wavelength filter.

In some embodiments, the different materials may include alternatinglayers of titanium dioxide (TiO₂) and silicon dioxide (SiO₂). Usingtitanium dioxide and silicon dioxide as the alternating layers in awavelength filter may reduce the overall thickness of the structure ofthe wavelength filter.

The stack of alternating layers may have any suitable thickness toprovide a desired level of filtering of excitation energy while allowingtransmission of luminescence. A wavelength filter may have a thicknessof approximately 3 microns, approximately 3.5 microns, approximately 4microns, or approximately 4.5 microns. A wavelength filter may be formedacross multiple pixels of an integrated device.

FIG. 7-5 shows a cross-sectional view of wavelength filter 7-500 of anintegrated device. Wavelength filter includes alternating layers oflayer 7-501 and layer 7-502. Layer 7-501 and layer 7-502 have differentindices of refraction. For example, layer 7-501 may have a higher indexof refraction than layer 7-502. Different indices of refraction forlayers 7-501 and 7-502 can be achieved by using materials that havedifferent indices of refraction. In some embodiments, layer 7-501 mayinclude silicon dioxide, and layer 7-502 may include titanium dioxide.Wavelength filter 7-500 is formed within pixel region 7-508 havingsensor 7-510. Wavelength filter 7-500 may be configured to allowtransmission of light having one characteristic wavelength, λ2, whilesubstantially reflecting a different characteristic wavelength, λ1. Amarker may emit luminescence having 2, which can pass through excitationfilter 7-500 and be detected by sensor 7-510. Excitation energy mayinclude λ1. In this manner, sensor 7-510 may detect a signalsubstantially relative to luminescence of a marker.

In an exemplary embodiment, a waveguide filter may include 49 layers ofalternating TiO₂ and SiO₂ with a layer of SiO₂ on either side of thestack of TiO₂ and SiO₂ layer. The total thickness of the entire stack isapproximately 3.876 microns. FIG. 7-6 plots transmittance as a functionof wavelength for p-polarization, average polarization, s-polarizationfor an incident angle of approximately 10 degrees and transmittance atan incident angle of approximately 0 degrees. The extinction loss mayvary significantly over a wavelength range of approximately 21 nm and beapproximately 30 dB at approximately 646 nm for an incident angle ofapproximately 10. The thickness variation for such a filter may be lessthan approximately 1% in order to reduce artifacts due to thicknessuniformity.

Altering the thicknesses of layers within a multi-layer stack of awavelength filter may change its transmission properties. Changing thethickness of one or more layers in a wavelength filters may alter thewavelength of light the filter transmits. Using such a technique,sections of filtering elements may transmit one wavelength of lightwhile other sections may transmit a different wavelength of light toform a multi-wavelength filter. A section of a multi-wavelength filtermay overlap with a sensor configured to receive light transmittedthrough the section. The stack of alternating layers of amulti-wavelength filter may have two sections where the thickness of thealternating layers is approximately a quarter of a wavelength of lightwith a spacer having a half wavelength between the two quarterwavelength sections. The spacer may act as a Fabry-Perot resonator. Thespacer may have a variable thickness such that it has a thickness in afirst region that is larger than a thickness in a second region. Thewavelength of light transmitted by the wavelength filter may vary acrossthe filter because of the variation in thickness of the spacer. Thefirst region may allow transmission of light having a first wavelength,and the second region may allow transmission of light having a secondwavelength where the first wavelength is longer than the secondwavelength. Additionally, the first region of the filter maysubstantially reflect light having the second wavelength, and the secondregion of the filter may substantially reflect light having the firstwavelength. In this manner, the wavelength filter may provide variationof the wavelength of transmitted light across the wavelength filterprovided by the variation in the thickness of the spacer. Suchmulti-wavelength filters may be used in embodiments where more than oneluminescence wavelength is detected. The multi-wavelength filters mayreflect excitation energy by greater than 10 dB.

While fabricating such a wavelength filter, the spacer may be formed tothe thickness for transmission of the longer wavelength of light andetched in the regions where the shorter wavelength of light istransmitted. The high refractive index layer in regions that transmitthe shorter wavelength of light may have a patterned etch region.

FIG. 7-7 shows a cross-sectional view of a multi-wavelength filter7-700. Pixel 7-707 includes region 7-708 and region 7-709 ofmulti-wavelength filter 7-700 and sensor 7-710 and sensor 7-711.Multi-wavelength filter 7-700 includes alternating layers of 7-701 and7-702 and spacer 7-706. Layer 7-701 and layer 7-702 have differentindices of refraction. For example, layer 7-701 may have a higher indexof refraction than layer 7-702. Different indices of refraction forlayers 7-701 and 7-702 can be achieved by using materials that havedifferent indices of refraction. In some embodiments, layer 7-701 mayinclude silicon dioxide, and layer 7-702 may include titanium dioxide.As shown in FIG. 7-7, spacer 7-706 has a variable thickness such thatregion 7-708 has a smaller thickness than region 7-709. By varying thethickness of spacer 7-706, the wavelength of light transmitted by filter7-700 varies from region 7-708 to region 7-709. Depending on the region,the thickness of spacer 7-706 may equal approximately half thewavelength of the transmitted light. Spacer 7-706 has a smallerthickness in region 7-708 than in region 7-709 such that transmission oflight through region 7-708 has a smaller wavelength than transmission oflight through region 7-709. Variation in thickness of region 7-708 and7-709 may be approximately 5 nm, approximately 10 nm, approximately 15,approximately 20 nm, or approximately 25 nm. As shown in FIG. 7-7,region 7-708 allows transmission of λ1, and region 7-709 allowstransmission of λ2 where λ2 is larger than λ1. In this manner, sensor7-710 positioned in region 7-708 may detect λ1 and sensor 7-711positioned in region 7-709 may detect λ2. Additionally, multi-wavelengthfilter 7-706 may be configured to reflect excitation energy by bothregions 7-708 and 7-709. As shown in FIG. 7.7, both regions 7-708 and7-709 reflect λ3, which may have a longer wavelength than both λ1 andλ2.

FIG. 7-8 illustrates a plot of the transmittance of polarized light as afunction of wavelength of an exemplary wavelength filter having 13layers alternating TiO₂ and SiO₂, with a layer of SiO₂ on either side ofthe stack and a TiO₂ spacer having variable thickness. The alternatinglayers of TiO₂ and SiO₂ that form quarter wavelength layers havethicknesses of 76.20 nm and 118.51, respectively. The TiO₂ spacer has athickness in one region that is 137.16 nm and a thickness in anotherregion that is approximately 122.16 nm. The total thickness of theentire stack is 1.3 microns. The spacer layer has an etched region thatis approximately 15 nm thinner for the shorter wavelength bandpasstransmission regions of the filter than the longer wavelength bandpasstransmission regions. The transmission regions are centered atapproximately 660 nm and 685 nm. Additional cavity spacers may broadenthe transmission peaks to be squarer. The extinction loss may beapproximately in the range of 10 dB to 15 dB. The thickness variationfor such a filter may be less than approximately 1% in order to reduceartifacts due to thickness uniformity.

F. Sensor

Any suitable sensor capable of acquiring time bin information may beused for measurements to detect lifetimes of luminescent markers. Forexample, U.S. Provisional Patent Application 62/164,506, entitled“INTEGRATED DEVICE FOR TEMPORAL BINNING OF RECEIVED PHOTONS,” filed May20, 2015, describes a sensor capable of determining an arrival time of aphoton, and is incorporated by reference in its entirety. The sensorsare aligned such that each sample well has at least one sensor region todetect luminescence from the sample well. In some embodiments, theintegrated device may include Geiger mode avalanche photodiode arraysand/or single photon avalanche diode arrays (SPADs).

Described herein is an integrated photodetector that can accuratelymeasure, or “time-bin,” the timing of arrival of incident photons, andwhich may be used in a variety of applications, such as sequencing ofnucleic acids (e.g., DNA sequencing), for example. In some embodiments,the integrated photodetector can measure the arrival of photons withnanosecond or picosecond resolution, which can facilitate time-domainanalysis of the arrival of incident photons.

Some embodiments relate to an integrated circuit having a photodetectorthat produces charge carriers in response to incident photons and whichis capable of discriminating the timing at which the charge carriers aregenerated by the arrival of incident photons with respect to a referencetime (e.g., a trigger event). In some embodiments, a charge carriersegregation structure segregates charge carriers generated at differenttimes and directs the charge carriers into one or more charge carrierstorage regions (termed “bins”) that aggregate charge carriers producedwithin different time periods. Each bin stores charge carriers producedwithin a selected time interval. Reading out the charge stored in eachbin can provide information about the number of photons that arrivedwithin each time interval. Such an integrated circuit can be used in anyof a variety of applications, such as those described herein.

An example of an integrated circuit having a photodetection region and acharge carrier segregation structure will be described. In someembodiments, the integrated circuit may include an array of pixels, andeach pixel may include one or more photodetection regions and one ormore charge carrier segregation structures, as discussed below.

FIG. 7-9A shows a diagram of a pixel 7-900, according to someembodiments. Pixel 7-900 includes a photon absorption/carrier generationregion 7-902 (also referred to as a photodetection region), a carriertravel/capture region 7-906, a carrier storage region 7-908 having oneor more charge carrier storage regions, also referred to herein as“charge carrier storage bins” or simply “bins,” and readout circuitry7-910 for reading out signals from the charge carrier storage bins.

The photon absorption/carrier generation region 7-902 may be a region ofsemiconductor material (e.g., silicon) that can convert incident photonsinto photogenerated charge carriers. The photon absorption/carriergeneration region 7-902 may be exposed to light, and may receiveincident photons. When a photon is absorbed by the photonabsorption/carrier generation region 7-902 it may generatephotogenerated charge carriers, such as an electron/hole pair.Photogenerated charge carriers are also referred to herein simply as“charge carriers.”

An electric field may be established in the photon absorption/carriergeneration region 7-902. In some embodiments, the electric field may be“static,” as distinguished from the changing electric field in thecarrier travel/capture region 7-906. The electric field in the photonabsorption/carrier generation region 7-902 may include a lateralcomponent, a vertical component, or both a lateral and a verticalcomponent. The lateral component of the electric field may be in thedownward direction of FIG. 7-9A, as indicated by the arrows, whichinduces a force on photogenerated charge carriers that drives themtoward the carrier travel/capture region 106. The electric field may beformed in a variety of ways.

In some embodiments, one or more electrodes may be formed over thephoton absorption/carrier generation region 7-902. The electrodes(s) mayhave voltages applied thereto to establish an electric field in thephoton absorption/carrier generation region 7-902. Such electrode(s) maybe termed “photogate(s).” In some embodiments, photon absorption/carriergeneration region 7-902 may be a region of silicon that is fullydepleted of charge carriers.

In some embodiments, the electric field in the photon absorption/carriergeneration region 7-902 may be established by a junction, such as a PNjunction. The semiconductor material of the photon absorption/carriergeneration region 7-902 may be doped to form the PN junction with anorientation and/or shape that produces an electric field that induces aforce on photogenerated charge carriers that drives them toward thecarrier travel/capture region 7-906. In some embodiments, the P terminalof the PN junction diode may connected to a terminal that sets itsvoltage. Such a diode may be referred to as a “pinned” photodiode. Apinned photodiode may promote carrier recombination at the surface, dueto the terminal that sets its voltage and attracts carriers, which canreduce dark current. Photogenerated charge carriers that are desired tobe captured may pass underneath the recombination area at the surface.In some embodiments, the lateral electric field may be established usinga graded doping concentration in the semiconductor material.

As illustrated in FIG. 7-9A, a photon may be captured and a chargecarrier 7-901A (e.g., an electron) may be produced at time t1. In someembodiments, an electrical potential gradient may be established alongthe photon absorption/carrier generation region 7-902 and the carriertravel/capture region 7-906 that causes the charge carrier 7-901A totravel in the downward direction of FIG. 7-9A (as illustrated by thearrows shown in FIG. 7-9A). In response to the potential gradient, thecharge carrier 7-901A may move from its position at time t1 to a secondposition at time t2, a third position at time t3, a fourth position attime t4, and a fifth position at time t5. The charge carrier 7-901A thusmoves into the carrier travel/capture region 7-906 in response to thepotential gradient.

The carrier travel/capture region 7-906 may be a semiconductor region.In some embodiments, the carrier travel/capture region 7-906 may be asemiconductor region of the same material as photon absorption/carriergeneration region 7-902 (e.g., silicon) with the exception that carriertravel/capture region 7-906 may be shielded from incident light (e.g.,by an overlying opaque material, such as a metal layer).

In some embodiments, and as discussed further below, a potentialgradient may be established in the photon absorption/carrier generationregion 7-902 and the carrier travel/capture region 7-906 by electrodespositioned above these regions. However, the techniques described hereinare not limited as to particular positions of electrodes used forproducing an electric potential gradient. Nor are the techniquesdescribed herein limited to establishing an electric potential gradientusing electrodes. In some embodiments, an electric potential gradientmay be established using a spatially graded doping profile. Any suitabletechnique may be used for establishing an electric potential gradientthat causes charge carriers to travel along the photonabsorption/carrier generation region 7-902 and carrier travel/captureregion 7-906.

A charge carrier segregation structure may be formed in the pixel toenable segregating charge carriers produced at different times. In someembodiments, at least a portion of the charge carrier segregationstructure may be formed over the carrier travel/capture region 7-906. Aswill be described below, the charge carrier segregation structure mayinclude one or more electrodes formed over the carrier travel/captureregion 7-906, the voltage of which may be controlled by controlcircuitry to change the electric potential in the carrier travel/captureregion 7-906.

The electric potential in the carrier travel/capture region 7-906 may bechanged to enable capturing a charge carrier. The potential gradient maybe changed by changing the voltage on one or more electrodes overlyingthe carrier travel/capture region 7-906 to produce a potential barrierthat can confine a carrier within a predetermined spatial region. Forexample, the voltage on an electrode overlying the dashed line in thecarrier travel/capture region 7-906 of FIG. 7-9A may be changed at timet5 to raise a potential barrier along the dashed line in the carriertravel/capture region 7-906 of FIG. 7-9A, thereby capturing chargecarrier 7-901A. As shown in FIG. 7-9A, the carrier captured at time t5may be transferred to a bin “bin0” of carrier storage region 7-908. Thetransfer of the carrier to the charge carrier storage bin may beperformed by changing the potential in the carrier travel/capture region7-906 and/or carrier storage region 7-908 (e.g., by changing the voltageof electrode(s) overlying these regions) to cause the carrier to travelinto the charge carrier storage bin.

Changing the potential at a certain point in time within a predeterminedspatial region of the carrier travel/capture region 7-906 may enabletrapping a carrier that was generated by photon absorption that occurredwithin a specific time interval. By trapping photogenerated chargecarriers at different times and/or locations, the times at which thecharge carriers were generated by photon absorption may bediscriminated. In this sense, a charge carrier may be “time binned” bytrapping the charge carrier at a certain point in time and/or spaceafter the occurrence of a trigger event. The time binning of a chargecarrier within a particular bin provides information about the time atwhich the photogenerated charge carrier was generated by absorption ofan incident photon, and thus likewise “time bins,” with respect to thetrigger event, the arrival of the incident photon that produced thephotogenerated charge carrier.

FIG. 7-9B illustrates capturing a charge carrier at a different point intime and space. As shown in FIG. 7-9B, the voltage on an electrodeoverlying the dashed line in the carrier travel/capture region 7-906 maybe changed at time t9 to raise a potential barrier along the dashed linein the carrier travel/capture region 106 of FIG. 7-9B, thereby capturingcarrier 7-901B. As shown in FIG. 7-9B, the carrier captured at time t9may be transferred to a bin “bin1” of carrier storage region 7-908.Since charge carrier 7-901B is trapped at time t9, it represents aphoton absorption event that occurred at a different time (i.e., timet6) than the photon absorption event (i.e., at t1) for carrier 7-901A,which is captured at time t5.

Performing multiple measurements and aggregating charge carriers in thecharge carrier storage bins of carrier storage region 7-908 based on thetimes at which the charge carriers are captured can provide informationabout the times at which photons are captured in the photonabsorption/carrier generation area 7-902. Such information can be usefulin a variety of applications, as discussed above.

In some embodiments, the duration of time each time bin captures afteran excitation pulse may vary. For example, shorter time bins may be usedto detect luminescence shortly after the excitation pulse, while longertime bins may be used at times further from an excitation pulse. Byvarying the time bin intervals, the signal to noise ratio formeasurements of the electrical signal associated with each time bin maybe improved for a given sensor. Since the probability of a photonemission event is higher shortly after an excitation pulse, a time binwithin this time may have a shorter time interval to account for thepotential of more photons to detect. While at longer times, theprobability of photon emission may be less and a time bin detectingwithin this time may be longer to account for a potential fewer numberof photons. In some embodiments, a time bin with a significantly longertime duration may be used to distinguish among multiple lifetimes. Forexample, the majority of time bins may capture a time interval in therange of approximately 0.1-0.5 ns, while a time bin may capture a timeinterval in the range of approximately 2-5 ns. The number of time binsand/or the time interval of each bin may depend on the sensor used todetect the photons emitted from the sample object. Determining the timeinterval for each bin may include identifying the time intervals neededfor the number of time bins provided by the sensor to distinguish amongluminescent markers used for analysis of a sample. The distribution ofthe recorded histogram may be compared to known histograms of markersunder similar conditions and time bins to identify the type of marker inthe sample well. Different embodiments of the present application maymeasure lifetimes of markers but vary in the excitation energies used toexcite a marker, the number of sensor regions in each pixel, and/or thewavelength detected by the sensors.

III. Excitation Source

According to some embodiments, one or more excitation sources may belocated external to the integrated device, and may be arranged todeliver pulses of light to an integrated device having sample wells. Forexample, U.S. Provisional Patent Application 62/164,485, entitled“PULSED LASER,” filed May 20, 2015 describes a pulsed laser source thatmay be used as an excitation source, and is incorporated by reference inits entirety. The pulses of light may be coupled to a plurality ofsample wells and used to excite one or more markers within the wells,for example. The one or more excitation sources may deliver pulses oflight at one or more characteristic wavelengths, according to someimplementations. In some cases, an excitation source may be packaged asan exchangeable module that mounts in or couples to a base instrument,into which the integrated device may be loaded. Energy from anexcitation source may be delivered radiatively or non-radiatively to atleast one sample well or to at least one sample in at least one samplewell. In some implementations, an excitation source having acontrollable intensity may be arranged to deliver excitation energy to aplurality of pixels of an integrated device. The pixels may be arrangedin a linear array (e.g., row or column), or in a 2D array (e.g., asub-area of the array of pixels or the full array of pixels).

Any suitable light source may be used for an excitation source. Someembodiments may use incoherent light sources and other embodiments mayuse coherent light sources. By way of non-limiting examples, incoherentlight sources according to some embodiments may include different typesof light emitting diodes (LEDs) such as organic LEDs (OLEDs), quantumdots (QLEDs), nanowire LEDs, and (in)organic semiconductor LEDs. By wayof non-limiting examples, coherent light sources according to someembodiments may include different types of lasers such as semiconductorlasers (e.g., vertical cavity surface emitting lasers (VCSELs), edgeemitting lasers, and distributed-feedback (DFB) laser diodes).Additionally or alternatively, slab-coupled optical waveguide laser(SCOWLs) or other asymmetric single-mode waveguide structures may beused. In some implementations, coherent light sources may compriseorganic lasers, quantum dot lasers, and solid state lasers (e.g., aNd:YAG or ND:Glass laser, pumped by laser diodes or flashlamps). In someembodiments, a laser-diode-pumped fiber laser may be used. A coherentlight source may be passively mode locked to produce ultrashort pulses.There may be more than one type of excitation source for an array ofpixels on an integrated device. In some embodiments, different types ofexcitation sources may be combined. An excitation source may befabricated according to conventional technologies that are used tofabricate a selected type of excitation source.

By way of introduction and without limiting the invention, an examplearrangement of a coherent light source is depicted in FIG. 8-0A. Thedrawing illustrates an analytical instrument 8-100 that may include anultrashort-pulsed laser excitation source 8-110 as the excitationsource. The ultrashort pulsed laser 8-110 may comprise a gain medium8-105 (which may be a solid-state material is some embodiments), a pumpsource for exciting the gain medium (not shown), and at least two cavitymirrors 8-102, 8-104 that define ends of an optical laser cavity. Insome embodiments, there may be one or more additional optical elementsin the laser cavity for purposes of beam shaping, wavelength selection,and/or pulse forming. When operating, the pulsed-laser excitation source8-110 may produce an ultrashort optical pulse 8-120 that circulatesback-and-forth in the laser cavity between the cavity's end mirrors8-102, 8-104 and through the gain medium 8-105. One of the cavitymirrors 8-104 may partially transmit a portion of the circulating pulse,so that a train of optical pulses 8-122 is emitted from the pulsed laser8-110 to subsequent component 8-160, such as an optical component andintegrated device. The emitted pulses may sweep out a beam (indicated bythe dashed lines) that is characterized by a beam waist w.

Measured temporal intensity profiles of the emitted pulses 8-122 mayappear as depicted in FIG. 8-0B. In some embodiments, the peak intensityvalues of the emitted pulses may be approximately equal, and theprofiles may have a Gaussian temporal profile, though other profilessuch as a sech profile may be possible. In some cases, the pulses maynot have symmetric temporal profiles and may have other temporal shapes.In some embodiments, gain and/or loss dynamics may yield pulses havingasymmetric profiles. The duration of each pulse may be characterized bya full-width-half-maximum (FWHM) value, as indicated in FIG. 8-0B.Ultrashort optical pulses may have FWHM values less than 100picoseconds.

The pulses emitting from a laser excitation source may be separated byregular intervals T. In some embodiments, T may be determined by activegain and/or loss modulation rates in the laser. For mode-locked lasers,T may be determined by a round-trip travel time between the cavity endmirrors 8-102, 8-104. According to some embodiments, the pulseseparation time T may be between about 1 ns and about 100 ns. In somecases, the pulse separation time T may be between about 0.1 ns and about1 ns. In some implementations, the pulse separation time T may bebetween about 100 ns and about 2 μs.

In some embodiments, an optical system 8-140 may operate on a beam ofpulses 8-122 from a laser excitation source 8-110. For example, theoptical system may include one or more lenses to reshape the beam and/orchange the divergence of the beam. Reshaping of the beam may includeincreasing or decreasing the value of the beam waist and/or changing across-sectional shape of the beam (e.g., elliptical to circular,circular to elliptical, etc.). Changing the divergence of the beam maycomprise converging or diverging the beam flux. In some implementations,the optical system 8-140 may include an attenuator or amplifier tochange the amount of beam energy. In some cases, the optical system mayinclude wavelength filtering elements. In some implementations, theoptical system may include pulse shaping elements, e.g., a pulsestretcher and/or pulse compressor. In some embodiments, the opticalsystem may include one or more nonlinear optical elements, such as asaturable absorber for reducing a pulse length. According to someembodiments, the optical system 8-140 may include one or more elementsthat alter the polarization of pulses from a laser excitation source8-110.

In some implementations, an optical system 8-140 may include a nonlinearcrystal for converting the output wavelength from an excitation source8-110 to a shorter wavelength via frequency doubling or to a longerwavelength via parametric amplification. For example, an output of thelaser may be frequency-doubled in a nonlinear crystal (e.g., inperiodically-poled lithium niobate (PPLN)) or other non-poled nonlinearcrystal. Such a frequency-doubling process may allow more efficientlasers to generate wavelengths more suitable for excitation of selectedfluorophores.

The phrase “characteristic wavelength” or “wavelength” may refer to acentral or predominant wavelength within a limited bandwidth ofradiation produced by an excitation source. In some cases, it may referto a peak wavelength within a bandwidth of radiation produced by anexcitation source. A characteristic wavelength of an excitation sourcemay be selected based upon a choice of luminescent markers or probesthat are used in a bioanalysis device, for example. In someimplementations, the characteristic wavelength of a source of excitationenergy is selected for direct excitation (e.g., single photonexcitation) of a chosen fluorophore. In some implementations, thecharacteristic wavelength of an excitation source is selected forindirect excitation (e.g., multi-photon excitation or harmonicconversion to a wavelength that will provide direct excitation). In someembodiments, excitation radiation may be generated by a light sourcethat is configured to generate excitation energy at a particularwavelength for application to a sample well. In some embodiments, acharacteristic wavelength of the excitation source may be less than acharacteristic wavelength of corresponding emission from the sample. Forexample, an excitation source may emit radiation having a characteristicwavelength between 500 nm and 700 nm (e.g., 515 nm, 532 nm, 563 nm, 594nm, 612 nm, 632 nm, 647 nm). In some embodiments, an excitation sourcemay provide excitation energy centered at two different wavelengths,such as 532 nm and 593 nm for example.

In some embodiments, a pulsed excitation source may be used to excite aluminescent marker in order to measure an emission lifetime of theluminescent marker. This can be useful for distinguishing luminescentmarkers based on emission lifetime rather than emission color orwavelength. As an example, a pulsed excitation source may periodicallyexcite a luminescent marker in order to generate and detect subsequentphoton emission events that are used to determine a lifetime for themarker. Lifetime measurements of luminescent markers may be possiblewhen the excitation pulse from an excitation source transitions from apeak pulse power or intensity to a lower (e.g., nearly extinguished)power or intensity over a duration of time that is less than thelifetime of the luminescent marker. It may be beneficial if theexcitation pulse terminates quickly, so that it does not re-excite theluminescent marker during a post-excitation phase when a lifetime of theluminescent marker is being evaluated. By way of example and notlimitation, the pulse power may drop to approximately 20 dB,approximately 40 dB, approximately 80 dB, or approximately 120 dB lessthan the peak power after 250 picoseconds. In some implementations, thepulse power may drop to approximately 20 dB, approximately 40 dB,approximately 80 dB, or approximately 120 dB less than the peak powerafter 100 picoseconds.

An additional advantage of using ultrashort excitation pulses to exciteluminescent markers is to reduce photo-bleaching of the markers.Applying continuous excitation energy to a marker may bleach and/ordamage a luminescent marker over time. Even though a peak pulse power ofthe excitation source may be considerably higher than a level that wouldrapidly damage a marker at continuous exposure, the use of ultrashortpulses may increase the amount of time and number of useful measurementsbefore the marker becomes damaged by the excitation energy.

When using a pulsed excitation source to discern lifetimes ofluminescent markers, the time between pulses of excitation energy may beas long as or longer than a longest lifetime of the markers in order toobserve and evaluate emission events after each excitation pulse. Forexample, the time interval T (see FIG. 8-0B) between excitation pulsesmay be longer than any emission lifetime of the examined fluorophores.In this case, a subsequent pulse may not arrive before an excitedfluorophore from a previous pulse has had a reasonable amount of time tofluoresce. In some embodiments, the interval T needs to be long enoughto determine a time between an excitation pulse that excites afluorophore and a subsequent photon emitted by the fluorophore aftertermination of excitation pulse and before the next excitation pulse.

Although the interval between excitation pulses T should be long enoughto observe decay properties of the fluorophores, it is also desirablethat T is short enough to allow many measurements to be made in a shortperiod of time. By way of example and not limitation, emission lifetimesof fluorophores used in some applications may be in the range of about100 picoseconds to about 10 nanoseconds. Accordingly, excitation pulsesused to detect and/or discern such lifetimes may have durations (FWHM)ranging from about 25 picoseconds to about 2 nanoseconds, and may beprovided at pulse repetition rates ranging from about 20 MHz to about 1GHz.

In further detail, any suitable techniques for modulating the excitationenergy to create a pulsed excitation source for lifetime measurementsmay be used. Direct modulation of an excitation source, such as a laser,may involve modulating the electrical drive signal of the excitationsource so the emitted power is in the form of pulses. The input powerfor a light source, including the optical pumping power, andexcited-state carrier injection and/or carrier removal from a portion ofthe gain region, may be modulated to affect the gain of the gain medium,allowing the formation of pulses of excitation energy through dynamicgain shaping. Additionally, the quality (Q) factor of the opticalresonator may be modulated by various means to form pulses usingQ-switching techniques. Such Q-switching techniques may be active and/orpassive. The longitudinal modes of a resonant cavity of a laser may bephase-locked to produce a series of pulses of emitted light throughmode-locking. Such mode-locking techniques may be active and/or passive.A laser cavity may include a separate absorbing section to allow formodulation of the carrier density and control of the absorption loss ofthat section, thus providing additional mechanisms for shaping theexcitation pulse. In some embodiments, an optical modulator may be usedto modulate a beam of continuous wave (CW) light to be in the form of apulse of excitation energy. In other embodiments, a signal sent to anacoustic optic modulator (AOM) coupled to an excitation source may beused to change the deflection, intensity, frequency, phase, and/orpolarization of the outputted light to produce a pulsed excitationenergy. AOMs may also be used for continuous wave beam scanning,Q-switching, and/or mode-locking. Although the above techniques aredescribed for creating a pulsed excitation source, any suitable way toproduce a pulsed excitation source may be used for measuring lifetimesof luminescent markers.

In some embodiments, techniques for forming a pulsed excitation sourcesuitable for lifetime measurements may include modulation of an inputelectrical signal that drives photon emission. Some excitation sources(e.g., diode lasers and LEDs) convert an electrical signal, such as aninput current, into a light signal. The characteristics of the lightsignal may depend on characteristics of the electrical signal. Inproducing a pulsed light signal, the electrical signal may vary overtime in order to produce a variable light signal. Modulating theelectrical signal to have a specific waveform may produce an opticalsignal with a specific waveform. The electrical signal may have asinusoidal waveform with a certain frequency and the resulting pulses oflight may occur within time intervals related to the frequency. Forexample, an electrical signal with a 500 MHz frequency may produce alight signal with pulses every 2 nanoseconds. Combined beams produced bydistinct pulsed excitation sources, whether similar or different fromeach other, may have a relative path difference below 1 mm.

In some excitation sources, such as laser diodes, the electrical signalchanges the carrier density and photons are produced through therecombination of electron and hole pairs. The carrier density is relatedto the light signal such that when the carrier density is above athreshold, a substantial number of coherent photons are generated viastimulated emission. Current supplied to a laser diode may injectelectrons or carriers into the device, and thereby increase the carrierdensity. When the carrier density is above a threshold, photons may begenerated at a faster rate than the current supplying the carriers, andthus the carrier density may decrease below the threshold and photongeneration reduces. With the photon generation reduced, the carrierdensity begins to increase again, due to continued current injection andthe absorption of photons, and eventually increases above the thresholdagain. This cycle leads to oscillations of the carrier density aroundthe threshold value for photon generation, resulting in an oscillatinglight signal. These dynamics, known as relaxation oscillations, can leadto artifacts in the light signal due to oscillations of the carrierdensity. When current is initially supplied to a laser, there may beoscillations before the light signal reaches a stable power due tooscillations in the carrier density. When forming a pulsed excitationsource, oscillations of the carrier density may introduce artifacts fora pulsed light signal. For example, the plot in FIG. 8-1 illustrates howthe carrier density may have relaxation oscillations and a correspondinglight signal with oscillating power through modulation by gainswitching. Artifacts from such relaxation oscillations may broaden apulsed light signal and/or produce a tail in the light signal, limitingthe lifetimes that can be detected by such a pulsed light source sincethe excitation signal may overlap with emitted photons by a luminescentmarker.

In some embodiments, techniques for shortening the time duration of anexcitation pulse may be used to reduce the excitation energy required todetect luminescent markers and thereby reduce or delay bleaching andother damage to the luminescent markers. Techniques for shortening thetime duration of the excitation pulse may be used to reduce the powerand/or intensity of the excitation energy after a maximum value or peakof the excitation pulse, allowing the detection of shorter lifetimes.Such techniques may electrically drive the excitation source in order toreduce the excitation power after the peak power. This may suppress atail of the pulse as shown in FIG. 8-2. An electrical driving signal maybe tailored to drive the intensity of the pulse of excitation energy tozero as quickly as possible after the peak pulse. An example of atailored electrical driving signal combined with gain switching is shownin FIG. 8-2. Such a technique may involve reversing the sign of anelectrical driving signal after the peak power is produced. Such atailored electrical driving signal may produce an optical output shownin FIG. 8-2. The electrical signal may be tailored to quickly reduce thecarrier density after the first relaxation oscillation or firstoscillation of the optical signal. By reducing the carrier density afterthe first oscillation, a light pulse of just the first oscillation maybe generated. The electrical signal may be configured to generate ashort pulse that turns the light signal off quickly by reducing thenumber of photons emitted after a peak in the signal, such as shown bythe plot in FIG. 8-3 showing the optical output of such an electricalsignal. A picosecond laser diode system may be designed to emit lightpulses, according to some embodiments. FIG. 8-4 illustrates a plot of anexemplary light pulse with a peak of 985 mW, a width of 84. 3picoseconds, and a signal reduced by approximately 24.3 dB approximately250 picoseconds after the peak. In some embodiments, saturableabsorbers, including semiconductor saturable absorbers (SESAMs) may beused to suppress the optical tail. In such embodiments, using thesaturable absorbers may suppress the optical tail by 3-5 dB or, in someinstances, greater than 5 dB. Reducing the effects of a tail in theexcitation pulse may reduce and/or eliminate any requirements onadditional filtering of the excitation energy, increase the range oflifetimes that can be measured, and/or enable faster pulse rates.Increasing the excitation pulse rate may enable more experiments to beconducted in a given time, which may decrease the time needed to acquireenough statistics to identify a lifetime for a marker labeling a sampleobject.

Additionally, two or more of these techniques may be used together togenerate pulsed excitation energy. For example, pulsed excitation energyemitted from a directly modulated source may be further modified usingoptical modulation techniques. Techniques for modulating the excitationpulse and tailoring the electrical pulse driving signal may be combinedin any suitable way to optimize a pulsed excitation energy forperforming lifetime measurements. A tailored electrical drive signal maybe applied to a pulsed excitation energy from a directly modulatedsource.

In some embodiments, a laser diode having a certain number of wire bondsmay be used as a pulsed excitation source. Laser diodes with more wirebonds may reduce the inductance of the excitation source. Laser diodeshaving a lower inductance may enable the current into the laser tooperate at a higher frequency. As shown in FIG. 8-5, when driven by an18V pulse in a 50 ohm transmission line, the laser source with 3 ohmseries resistance and 36 wire bonds has a higher current at higherfrequencies than the laser sources with fewer wire bonds. Selecting apackaging method to minimize inductance may improve the power suppliedto the excitation source at higher frequencies, enabling shorterexcitation pulses, faster reductions of optical power after the peak,and/or increased pulse repetition rate for detecting luminescentmarkers.

In some embodiments, a transmission line in combination with anexcitation source may be used for generating light pulses. Thetransmission line may match the impedance of a laser diode in order toimprove performance and/or quality of light pulses. In some embodiments,the transmission line impedance may be 50 ohms. In some instances, theterminating resistance may be similar to the resistance of the line inorder to avoid reflections. Alternatively or additionally, theterminating impedance may be similar to the impedance of the line inorder to avoid reflections. The terminating impedance may less than theimpedance of the line in order to reflect a negative pulse. In otherembodiments, the terminating impedance may have a capacitive orinductive component in order to control the shape of the negativereflection pulse. In other embodiments, the transmission line may allowfor a higher frequency of pulses. FIG. 8-6A illustrates an exampleprototype of a transmission line pulsar, and FIG. 8-6B illustratesexemplary light pulses obtained with such a transmission line. Using atransmission line may produce electrical pulses having a frequencywithin a range of 40 MHz to 500 MHz. A transmission line may be used incombination with a tailored electrical signal described above in orderto produce a pulsed light source with light pulses having a certain timeduration and a specific time interval.

Techniques for tailoring the electrical signal to improve the productionof light pulses may include connecting the excitation source to acircuit with a negative bias capability. In some embodiments, a negativebias may be provided on an excitation source after a light pulse emitsto reduce emission of a tail in the light pulse. FIG. 8-7 illustrates anexemplary circuit containing a current source, diode laser, resistor,capacitor, and switch that may be implemented to reduce the presence ofa tail in a light pulse. Such a circuit may create a constant currentthat bypasses the diode laser when the switch is closed, or in aconducting state. When the switch is open, the switch may have a highresistance and current may flow through the diode laser. Light pulsesmay be generated by opening and closing the switch to provideintermittent current to the diode laser. In some instances, the resistormay be sufficiently high and the capacitor sufficiently small such thatthere is a voltage across the capacitor when the switch is open and thediode laser emits light. When the switch is closed, the voltage acrossthe capacitor will reverse bias the diode laser. Such a reverse bias mayreduce or eliminate the presence of a tail in the light pulse. In suchinstances, the switch may be configured to close after the peak of thelight pulse in order to reduce the laser power shortly after the peaklight pulse. The value of the resistor in the circuit may be selectedsuch that the charge on the capacitor will discharge before the switchis subsequently opened and/or a subsequent light pulse is generated bythe laser diode.

Additional circuit components may be provided to tailor an electricalsignal of a laser diode in order to produce light pulses. In someembodiments, multiple capacitors, resistors, and voltages may beconnected as a network circuit to control the waveform of an electricalsignal supplied to a laser diode. A controlled waveform may be createdby switching a number of voltages, V1, V2, . . . , VN with correspondingsignals S1, S2, . . . , SN when there are N capacitor sub-circuits. Anexemplary network circuit of four capacitor sub-circuits is shown inFIG. 8-8 where a controlled electrical waveform may be created byswitching voltages V1, V2, V3, and V4 with signals S1, S2, S3, and S4,respectively. In some embodiments, voltages V1, V2, V3, and V4 may bevariable. In the example shown in FIG. 8-8, V4 is negative to the laserand may create a reverse bias depending on signal S4. Timing of thefrequency of light pulses emitted by the laser, the duration of eachlight pulse, and features of each light pulse may be adjusted with thesignal inputs S1, S2, S3, and S4. In some embodiments, additionalresistance may be added to lower the peak current. In such instances,the resistance may be added after one or more of the switches S1, S2,S3, S4. Although FIG. 8-8 shows one configuration with four capacitorsand four voltages, any suitable configuration and any suitable number ofadditional circuit components may be provided to produce a tailoredelectrical signal to the laser diode to generate light pulses forlifetime measurements.

In some embodiments, an electrical signal for generating light pulsesmay use a circuit having discrete components, including radio frequency(RF) and/or microwave components. Discrete components that may beincluded in such a circuit are DC blocks, adaptors, logic gates,terminators, phase shifters, delays, attenuators, combiners, and/or RFamplifiers. Such components may be used to create a positive electricalsignal having a certain amplitude followed by a negative electricalsignal with another amplitude. There may be a delay between the positiveand negative electrical signals. FIG. 8-9A illustrates an exemplarycircuit having one RF amplifier that may be used to produce a tailoredelectrical signal as an output pulse, such as the pulse profile shown inFIG. 8-9B, that may be supplied to an excitation source, such as a laserdiode, to emit a light pulse. In other embodiments, a circuit mayproduce multiple electrical signals combined to form an electrical pulsesignal configured to drive an excitation source. Such a circuit mayproduce a differential output which may be used to increase the power ofthe light pulse. By adjusting the discrete components of the circuit,the electrical output signal may be adjusted to produce a light pulsesuitable for lifetime measurements. In an example shown in FIG. 8-10A,two RF amplifiers are used to produce an output pulse signal having aprofile shown in FIG. 8-10B consisting of a positive electrical signalpulse and a corresponding negative electrical signal pulse where thepositive and negative electrical signal pulses overlap and have asimilar width.

In some embodiments, excitation sources may be combined to generatelight pulses for lifetime measurements. Synchronized pulsed sources maybe coupled to a circuit or load over a certain distance. In someembodiments, excitation sources may be coupled in parallel to a circuit.The excitation sources may be from the same source or from multiplesources. In some embodiments with multiple sources, the multiple sourcesmay vary in type of excitation source. When combining sources, it may beimportant to consider the impedance of the circuit and the excitationsources in order to have sufficient power supplied to the excitationsources. Combination of sources may be achieved by using one or more ofthe above-described techniques for producing a pulsed excitation source.FIG. 8-11A illustrates a schematic for combining four different sourceshaving one or more impedance values. FIG. 8-11B shows a plot of current,power efficiency, and voltage as a function of impedance. This exemplaryembodiment shows 4 sources delivering power on 50 ohms transmissionlines and that optimal power delivery occurs when the impedance of theload equals the ratio of the impedance of the individual lines to thenumber of sources.

An excitation source may include a battery or any other power supplyarranged to provide power to the excitation source. For example, anexcitation source may be located in a base instrument and its operatingpower may be received through an integrated bioanalysis device to whichit is coupled (e.g., via conducting power leads). An excitation sourcemay be controlled independently of or in collaboration with control ofan integrated bioanalysis device. As just one example, control signalsfor an excitation source may be provided to the excitation sourcewirelessly or via a wired interconnection (e.g., a USB interconnect)with a personal computer and/or the integrated bioanalysis device.

In some implementations, an excitation source may be operated in atime-gated and/or synchronized manner with one or more sensors of anintegrated device. For example, an excitation source may be turned on toexcite a luminescent marker, and then turned off. The sensor may beturned off while the excitation source is turned on, and then may beturned on for a sampling interval after the excitation source is turnedoff. In some embodiments, the sensor may be turned on for a samplinginterval while the excitation source is turned on.

IV. Alignment of Excitation Source to Integrated Device

Positioning of excitation energy from one or more excitation sources toa grating coupler on an integrated device may occur using any suitabletechniques. In some embodiments, excitation energy may be directed froman excitation source through one or more optical components to a gratingcoupler. In such embodiments, the excitation energy may be projectedonto the integrated device. Alignment of such an excitation source mayoccur actively by positioning the excitation source and/or opticalcomponents. In other embodiments, an excitation source may be aligned toa grating coupler through components that position the excitation sourcerelative to the grating coupler. Positioning of such an excitationenergy source may occur through passive alignment of an optical fiberconfigured to provide excitation energy within a ferrule. In suchembodiments, the excitation source may be connected directly orindirectly to the integrated device.

A. Active Alignment

Alignment of one or more excitation sources to an integrated device mayoccur once before excitation energy is delivered to a sample well and/ormultiple times while the integrated device is used for an analysis. Anysuitable techniques for alignment and/or stabilization of one or moreexcitation sources may be used to couple excitation energy to theintegrated device. In some embodiments, the external excitation sourcemay be aligned to the integrated device and then fixed in positionwithout further adjustment throughout the duration while the integrateddevice is used for analysis. In other embodiments, a feedback mechanismis provided to improve the alignment of the excitation source to theintegrated device. The feedback mechanism may be used in combinationwith an operator using a manual alignment mechanism to align theexcitation source based on a feedback signal. In other instances, anautomatic alignment mechanism may automatically adjust the alignment ofthe excitation source to the integrated device based on a feedbacksignal. Automatic alignment may occur before operation of the integrateddevice occurs and/or before acquiring a measurement. Automatic alignmentmay also occur during operation of the integrated device whilemeasurements are acquired. In some instances, the time when automaticalignment occurs may correspond to times when the sensor is not activelyacquiring measurements. Readjusting the alignment of the excitationsource to the integrated device may improve stability of the excitationsource and/or consistency in sensor measurements over time among one ormore samples.

The alignment may be actively controlled in multiple dimensions based ona feedback signal. The multiple dimensions may include the x-y lateralpositions of the excitation energy beam to the grating coupler, theangle of incidence of the beam along the direction of the gratingcoupler, the z direction of focus of the beam, and/or an orthogonalangle of incidence of the beam perpendicular to the direction of thegrating coupler. The excitation source may be connected to theintegrated device through a frame mount. Such an alignment mechanism maybe performed using one or more servo-controlled optical elements. Theactive alignment mechanism may increase the overall coupling efficiencyof the excitation source to the one or more waveguides in the integrateddevice. Such an alignment mechanism may decrease additional costsassociated with a disposable integrated device as compared to otheralignment methods, and/or reduce the footprint on the integrated devicerequired by other alignment approaches. Additionally, an activealignment mechanism may reduce the training needed for an operator tooperate such an integrated device and/or system.

The components performing the alignment may depend on the configurationof the excitation source and the integrated device. In some embodiments,an integrated device and an excitation source may be configured intoseparate modules. A base instrument may include a dark box with a lidand a socket to connect the integrated device to electrical circuitry inorder to detect an electrical signal from the sensor on the integrateddevice. An excitation source module may include one or more excitationsources, socket connections for the one or more excitation sources,and/or optical components to direct light to the integrated device. Whena laser is used as an excitation source, the excitation source modulemay be referred to as a laser module.

In some embodiments, optical components couple the excitation source tothe integrated device in free space. The mirror and/or additionaloptical components may direct excitation energy from the excitationsource to the waveguide. Optical components may increase the depth offocus of the excitation source. FIG. 9-1 illustrates base instrument9-112 configured to receive integrated device 9-102. Base instrument9-112 includes mirror 9-114 arranged to direct excitation energy (shownas dashed lines) received from excitation source module 9-110 and directexcitation energy towards a region of integrated device 9-102, such as asurface proximate a grating coupler. Excitation source module 9-110includes excitation source 9-106 and adjustment mechanism 9-108.Alignment mechanism 9-108 may modulate the x, y, z positions, the x andy incidence angles, and the z positioning or focus of excitation source9-206 to align the excitation source and/or optical components in orderto improve coupling of the excitation energy to the integrated device.Such optical components may include mirrors, reflectors, lenses, prisms,gratings, and/or tiltable windows, such as the example set of opticalcomponents arranged in FIG. 9-3. Alignment of excitation source 9-106may be determined through feedback (shown as solid arrow) fromintegrated device 9-102 and received by excitation source module 9-110through socket 9-116 that couples to integrated device 9-102. Electricalsignals received by socket 9-116 may indicate the alignment ofexcitation source 9-106. Such electrical signals may be used in anactive feedback process by sending electrical signals received fromintegrated device 9-102 to excitation source module 9-110 and provide asignal to alignment mechanism 9-108 indicating adjustment of one or moreparameters of alignment mechanism 9-108 and/or excitation source 9-106.When the electrical signal is received by excitation source module9-110, alignment mechanism 9-108 may consist of adjusting the positionof excitation source 9-106 and/or optical components. Such an alignmentprocess may occur automatically and/or manually.

In some embodiments, a mirror is provided as part of the excitationsource module. FIG. 9-2 illustrates a schematic of excitation sourcemodule 9-210 that includes mirror 9-214 configured to direct excitationenergy (shown in dashed lines) to integrated device 9-202. Baseinstrument 9-212 is configured to receive integrated device 9-202 andexcitation source module 9-210 such that mirror 9-214 is positioned todirect excitation energy from excitation source 9-206 to integrateddevice 9-202. Alignment module 9-206 may modulate one or more parametersof excitation source 9-206 and/or mirror 9-214 based on a feedbacksignal (shown as solid arrow) received by excitation source module 9-210from socket 9-216 configured to couple to 9-202. The feedback signal mayprovide an indication of the alignment of excitation energy to a region(e.g., grating coupler) of integrated device 9-202. Alignment mechanism9-208 may modulate the x, y, z positions, the x and y incidence angles,and the z positioning or focus of excitation source 9-206 and/or opticalcomponents to improve coupling of the excitation energy to theintegrated device. Such optical components may include mirrors,reflectors, lenses, prisms, gratings, and/or tiltable windows, such asthe example set of optical components arranged in FIG. 9-3.

In some embodiments, excitation energy from the excitation source moduleis coupled to the base instrument through an optical fiber, such as inthe example shown in FIG. 9-4. The optical fiber connects an excitationenergy output from the excitation source model to an input in the baseinstrument. In some embodiments, the optical fiber is a single modefiber. Optical components within the base instrument direct theexcitation energy to the integrated device. Alignment of the excitationenergy to the integrated device may occur through such opticalcomponents. Feedback from an electrical signal received by theintegrated device may direct adjustment of one or more opticalcomponents within the base instrument to improve the alignment of theexcitation energy to the integrated device.

Another example of an excitation module that rests on top of integrateddevice is illustrated in FIGS. 9-5 to 9-11. The excitation source modulecontains one or more excitation sources in addition to optical andalignment components for delivering excitation energy to one or moregrating couplers on the integrated device positioned at 9-501. Alignmentcomponents are included in module 9-502 and are positioned overintegrated device 9-501. Coarse alignment of the excitation source tothe integrated device may occur by placing steel ball bearings on thesurface of the integrated device and forming, on the excitation sourcemodule, magnetized grooves that automatically register the steel ballbearings. As shown in FIG. 9-6, there are three steel ball bearings9-503 bonded to the integrated device surface 9-501 and the excitationsource module has three magnetized radial v-grooves 9-504 thatautomatically register the three ball bearings to stabilize positioningof the excitation source module to the integrated device, including allthree translational and all three rotational degrees of freedom. Theexcitation source module 9-502 may include a hinge pin 9-505 to tilt themodule away from integrated device in an up position to provide accessto the integrated device. FIG. 9-6 shows the excitation source module inthe up position. FIG. 9-7 shows the same excitation source module in thedown position when the steel ball bearings are registered by themagnetized grooves. On the top surface of the excitation source modulethere is a circuit board 9-506 with an excitation source, in thisexample a laser diode. The circuit board 9-506 with the laser diode,collimating lens 9-508, and heat sink 9-507 are shown in further detailin FIG. 9-8. The printed circuit board may have circuitry for drivingand supporting the laser diode. There may be a focusable collimatinglens to focus light from the laser diode. Additionally, there is a heatsink surrounding the laser diode for heat dissipation. The excitationsource module has an entrance port 9-509 for the collimated laser beam,as shown in FIG. 9-9. The entrance port is surrounded by a heatconducting block 9-510 to receive the laser diode heat sink.Additionally, rotary actuators 9-511 a, 9-511 b, and 9-511 c areprovided on the excitation source module to control the x-y position andincidence angle of the delivered beam to the integrated device. Therotary actuators may be any suitable actuator that produces a rotarymotion or torque, including stepper motors and servos. The rotaryactuators control plane parallel plates that act as tiltable windowsalong the light path. The tilt of each of the plates is controlled bythe corresponding rotary actuator. When using a point light source, suchas a laser diode, introducing a plane parallel plate before a focusinglens in the light path adjusts the angle of incidence while introducinga plane parallel plate after the focusing lens adjust the x-y positionof the beam. Optical components inside the excitation source moduledirect and modulate light from the laser diode, as illustrated in across-sectional view shown in FIG. 9-10. The path of the light beam isdenoted by dotted lines. The light beam enters from the top surface ofthe excitation source module, which is into the plane of view frombehind, and is reflected by a 45 degree prism to propagate to the top ofthe plane view of FIG. 9-10. A rotary actuator 9-511 a with a planeparallel plate 9-512 is provided along the light path before ananamorphic prism 9-513 with an adjustable angle of incidence. Theanamorphic prism stretches or shrinks the beam in one direction toadjust the cross-section of the beam from an elliptical profile to besubstantially more circular in profile. The amount of beam stretching orshrinking is controlled by the angle of incidence of the prism. Thelight beam then reflects off another prism 9-514 to pass through afocusing lens 9-515 to bring the beam to focus on the integrated device.The light beam then passes through two more rotary actuator driventiltable windows 9-516 that adjust the x-y position of the beam on theintegrated device and the light beam propagates perpendicularly out ofthe plane of view towards the viewer in FIG. 9-10. An additionalviewpoint illustrating the positioning of the three rotary actuatorswithin the excitation source module is shown in FIG. 9-11.

Alignment along the excitation energy path from an excitation source toan integrated device may be performed at any suitable time after anintegrated device is positioned within a base instrument. In someembodiments, the alignment of the excitation source and/or opticalcomponents in either the excitation source module or the base instrumentis initially obtained and there is no subsequent re-alignment process.Such an alignment procedure may be called an initial static alignment.An initial static alignment process may be manual, automated, or acombination of a manual and automated process. In some embodiments, thealignment of the excitation source and/or optical components may undergomultiple re-alignments in a continuous, active alignment process. Acontinuous active alignment process may be automatic based on a feedbackelectrical signal from the integrated device. In some embodiments, thealignment process may include an initial manual alignment followed by acontinuous active alignment.

Various possible parameters of the beam of excitation energy may beadjusted during an alignment process. Such parameters may include x-y-zpositions, focus, angle with respect to the waveguide, angleperpendicular to the waveguide, and/or the magnification, aspect ratio,and/or astigmatism content of the beam. Variations for projectedexcitation energy beam alignment to the integrated device are summarizedin FIG. 9-12.

B. Passive Alignment

Positioning of one or more excitation sources may occur through passivealignment of an optical fiber, which carries excitation energy, to anintegrated device. Passive alignment techniques may include a series ofcomponents that securely position the optical fiber with respect to thegrating coupler in order to couple excitation energy to a waveguide. Thecomponents are made of any suitable material, including plastic andmetal. Passive alignment components are any suitable size in order toaccommodate an optical fiber of a certain size. Additionally, thecomponents may be designed for ease of connecting and disconnecting anoptical fiber to an integrated device.

In some embodiments, some of the passive alignment components may form areceptacle for connecting and positioning of a ferrule for an opticalfiber which carries excitation energy and aligns the ferrule withrespect to an integrated device. The receptacle may be made of anysuitable material, such as plastic or metal. The receptacle may be sizedto frame the ferrule.

Passive alignment components may include mechanical reference componentsto achieve positioning of an optical fiber to an integrated device. Themechanical reference components may improve precision in positioning ofthe optical fiber with respect to a grating coupler on the integrateddevice. The mechanical reference components may be located on a surfaceof the integrated device. In some instances, mechanical referencescomponents set an angle of alignment of the ferrule to a surface of theintegrated device. Mechanical reference components may set a distancefrom the surface of the integrated device such than the fiber ferruledoes not contact the integrated device's surface. Additionally oralternatively, mechanical reference components may provide an indicatorfor a user to identify when correct alignment is achieved. As anexample, a mechanical reference may provide a visual indication ofcorrect alignment of the fiber ferrule within the receptacle. In someembodiments, a clip may be attached to the fiber ferrule to achievecorrect rotational positioning of the fiber ferrule with respect to oneor more passive alignment components.

Illustrated in FIGS. 9-12 to 9-16 are exemplary passive alignmentcomponents that may form a receptacle and/or mechanical references for afiber ferrule. The fiber ferrule has a ferrule wall 9-1201 and asingle-mode fiber (SMF) core 9-1202. In some embodiments, the fiberferrule may have a diameter of 0.5 mm. As shown in FIG. 9-12, a planarcross-section view of an exemplary receptacle may include a component9-1203 with two alignment walls to form a receptacle and contact thefiber ferrule when the fiber ferrule is positioned within thereceptacle. The alignment walls may form a corner with an angle of 90degrees. In some embodiments, one of the alignment walls may beoptionally angled at 5 degrees from perpendicular to the surface of theintegrated device. Mechanical reference components may position thefiber ferrule within the receptacle. In some embodiments, mechanicalreference components on the surface of the integrated device may includea first mechanical reference cylinder aligned with the corner of thereceptacle formed by the two alignment walls and a second mechanicalreference cylinder aligned with a side of the alignment wall component.The first mechanical reference cylinder 9-1205 may be termed “cornerpad” and the second mechanical reference cylinder 9-1204 may be termed“side pad.” The mechanical reference cylinders may be formed byelectroplating gold on the surface of the integrated device. Additionalmechanical reference components may include one or more z-stop pads9-1206 positioned on the surface of the integrated device to maintain adistance between the end of the fiber ferrule and the surface of theintegrated device. The z-stop pads may be formed by electroplating goldon the surface of the integrated device. In some embodiments, a moldedplastic clip 9-1207 may be attached to the fiber ferrule to providerotational alignment with respect to the alignment wall component toposition the ferrule into the corner of the two alignment walls byattaching to the alignment wall component. The molded plastic integrateddevice may also be configured to position the ferrule a distance fromthe surface of the integrated device, providing a z-stop when insertingthe ferrule into the receptacle.

FIG. 9-13 illustrates a cross-section along line A-A′ shown in FIG. 9-12of an embodiment of a passive receptacle where the fiber ferrule isaligned with respect to the integrated device surface 9-1208. The end ofthe fiber ferrule may be angled to 8 degrees perpendicular to thedirection of the fiber core. Such an angle may be formed by polishingthe end of the fiber ferrule. One or more z-stop pads and/or a cornerpad may be provided on the surface of the integrated device. Whilepositioned in the receptacle, the fiber core may overlap with thegrating coupler 9-1209 in the integrated device in order to coupleexcitation energy to a waveguide.

Another embodiment of alignment of the fiber ferrule is shown in FIG.9-14 illustrating a cross-section along line A-A′ shown in FIG. 9-12. Inthis embodiment, a molded plastic clip 9-1207 is attached to the fiberferrule 9-1201 to provide positioning of the fiber ferrule 9-1201 withrespect to the alignment wall component 9-1203 such that the ferrule isin contact with the alignment walls and forced into the 90 degree cornerbetween the two alignment walls. The molded plastic clip attached to thefiber ferrule may also provide a z-stop for the fiber ferrule by havinga section that extends from the end of the fiber ferrule such that whenthe molded plastic clip is in contact with the integrated device the endof the fiber ferrule is positioned a distance from the surface of theintegrated device.

In another embodiment, a step is formed on the metal surface of theintegrated device to provide a z-stop when positioning the fiberferrule. FIG. 9-15 illustrates a cross-section view of such anembodiment along line A-A′ of FIG. 9-12. As the fiber ferrule 9-1201 ispositioned, the end of the fiber ferrule contacts step 9-1210 andpositions the fiber ferrule with respect to the integrated devicesurface.

FIG. 9-16 illustrates an embodiment where one of the alignment walls9-1603 has a 5 degree angle with respect to perpendicular to theintegrated device surface. When positioned in such a receptacle, thefiber ferrule contacts the angled alignment wall such that the fibercore is also at an angle perpendicular to the integrated device surface.The end of the fiber ferrule may be perpendicular to the direction ofthe fiber core. In such an embodiment, the end of the fiber ferrule mayhave no angle polished since the angle of the optical fiber to theintegrated device surface is achieved by the angle of the alignmentwall.

In some embodiments, a fiber ferrule may be positioned in a moldedplastic component substantially parallel to the surface of theintegrated device and the molded plastic component may have one or morelenses to direct light. Excitation energy from such a fiber ferrule maypropagate from the optical fiber through the molded plastic component tocouple with a grating coupler on the integrated device surface. Themolded plastic component may be made from a material having an index ofrefraction to direct the excitation energy towards the grating coupler.Additionally, the molded plastic may be formed with edge angles todirect the excitation energy towards the grating coupler. Whenpositioned in the molded plastic component, the fiber ferrule may bealigned within the molded plastic component such that through acombination of the index of refraction and one or more edge angles ofthe molded plastic component the excitation energy may be directedtowards the grating coupler on the integrated device. FIG. 9-17illustrates an exemplary molded plastic component configured to fit afiber ferrule with a diameter of 500 microns. Excitation energy from theoptical fiber propagates through a collimating surface to an angledsurface and reflects from the angled surface, propagating through themolded plastic of a certain refractive index, to the grating coupler.The angle of incidence of the excitation energy at integrated devicesurface may depend on the angle of the angled surface and the index ofrefraction of the material. In some embodiments, the angled surface maybe angled at 45 degrees minus the ratio of the angle of incidence to theindex of refraction in order to align the excitation energy to theintegrated device.

FIG. 9-18 illustrates an exemplary arrangement of a fiber ferrule, analignment wall component, and an integrated device. The fiber ferrule ispositioned within the alignment wall component in a corner of theintegrated device. The fiber ferrule has a diameter of 0.5 mm. Thealignment walls are 0.75 mm thick. Alignment pads are positioned insidethe receptacle and are 200 microns in diameter. When positioned in sucha receptacle, excitation energy from the optical fiber may couple withan initial waveguide aligned in a direction of the integrated devicehaving a certain length. In some embodiments, the initial waveguide isaligned in the direction of the integrated device with a length ofapproximately 9 mm.

FIG. 9-19 illustrates coupling of the excitation energy from the fibercore to a waveguide via the grating coupler. In some embodiments, theexcitation energy beam may expand to 15 microns in diameter. The radiusof curvature of the grating coupler features may be approximately 81microns where the grating coupler refocuses the excitation beam. Thegrating coupler may then refocus excitation energy towards a horn andultimately a waveguide.

In some embodiments, passive alignment may occur through a beamprojected onto a coupler. Projecting a beam of excitation energy mayoccur when there is sufficient stability of the optical apparatus toallow proper alignment. During manufacture, alignment of a beam may beset such that when a integrated device is correctly positioned the beamcouples to the grating coupler on the integrated device. The integrateddevice may be inserted using techniques that require accurate alignmentof an inserted integrated device to a laser module that includes theexcitation source. As an example, alignment of an inserted integrateddevice may be achieved passively through the use of kinematic balls androds. In this case, balls, positioned on the integrated device, allowthe laser module to register the integrated device and further align theintegrated device to the laser module after the integrated device isplaced in proximity by the user. In some embodiments, the positionalaccuracy of the integrated device is within the beam diameter. As anexample, placing balls on a integrated device may allow positioning of a20 μm beam to an accuracy within 5 μm. In some instances, the angle ofthe beam to the grating coupler may have accuracy to within a fewmilliradians. Additionally, the spacing of the balls on the integrateddevice may be adjusted to allow for improved manual placement of theintegrated device with respect to the light module. As an example,spacing of the balls to have more than a 5 mm separation may allow formilliradian accuracy on manual placement. Such passive alignmenttechniques may lower the overall cost for the system and reduce a needfor active alignment techniques that may interfere with operation of thesystem.

C. Excitation Energy Monitoring Sensors

A variety of monitoring sensors may be formed within the integratedsubstrate and configured to monitor the intensity and/or power ofexcitation energy at different locations within the integrated device.An electrical signal produced by such monitoring sensors may be used ina feedback process during operation of the integrated device. Theelectrical signal from the monitoring signals may also indicate areduced quality of the excitation energy propagated through theintegrated device, producing an error signal for an operator. This mayinclude an indication that an excitation source has or is failing. Insome instances, the monitoring sensors may provide a feedback signalduring alignment of the excitation energy to the grating coupler. Themonitoring sensors may be any suitable sensor, including light sensorssuch as photodetectors and photodiodes.

Monitoring sensors may be provided to monitor the excitation energyalong a waveguide. Sensors may be provided at the beginning of thewaveguide where the excitation energy initially couples with thewaveguide, at the end of the waveguide after the waveguide has coupledwith a row of sample wells, and/or anywhere along the length of thewaveguide. A grating coupler may be formed to improve coupling of theexcitation energy out of the waveguide in order to be detected by asensor. The information from the monitoring sensors may be used tocontrol parts of the system and/or as inputs for signal processing.

In some embodiments, a monitoring sensor is positioned underneath thegrating coupler to receive excitation energy that passes through thegrating coupler. The signal based on the intensity of the excitationenergy detected by such a sensor may be used to position and align theexcitation energy beam to the grating coupler. In some instances,multiple monitoring sensors are located underneath the grating coupler.A non-transparent layer of material, such as metal, having multipleholes may be located between the grating coupler and the multiplesensors. Each hole in the non-transparent layer may overlap with eachsensor such that excitation energy that passes through the hole isdetected by the sensor. By detecting excitation energy that passesthrough each hole by the multiple sensors, the position of theexcitation energy beam on the grating may be determined. FIG. 9-20 showsan example of such an arrangement where four holes 9-2010 in a metallayer are positioned to let light pass to four sensors 9-2008, which maybe considered a quadrant photodetector. As excitation energy 9-2004couples to the grating coupler 9-2002 and into tapered waveguide 9-2006,some of the excitation energy may pass through the holes 9-2010 in themetal layer to one or more of sensors 9-2008. The metal layer may act asa reflecting layer in regions without holes 9-2010 and may improvecoupling of excitation energy 9-2004 to grating coupler 9-2002 andtapered waveguide 9-2006. The signal from the sensors 9-2008 may providea vector for alignment feedback, with sufficient information to deduceboth the magnitude and the sign required for each adjustment in thefeedback loop. In some instances, alignment feedback may be providedwith each pulse of excitation energy. Alignment may be achieved when thebeam of excitation energy is centered on the four sensors.

In some embodiments, one or more sensors associated with each pixel maybe used for monitoring and/or alignment of the excitation energy. Thepower or intensity of the excitation energy may be set to a low value,such as a ‘low-power’ mode and positioning of the excitation source maybe performed by adjusting one or more of the x-y-z positions and/orangles relative to the waveguide in the integrated device. Improvingalignment of the excitation energy may occur by positioning the beam ofexcitation energy where the intensity signal detected by thephotodetectors increases to a certain value. In some instances,adjusting parameters of the excitation source may be performed todetermine an intensity signal above a certain value, indicatingsufficient alignment for operation.

In some embodiments, monitor sensors may be located at the end of eachwaveguide to detect the amount of light propagated along each waveguide.A grating associated with each monitor sensor may be positioned suchthat the grating and monitor sensor are located on opposite sides of thewaveguide. An example of sensors 9-2101 a and 9-2101 b and gratings9-2102 a and 9-2102 b provided at both ends of a waveguide is shown inFIG. 9-21. In some instances, signals at both ends of the waveguide maybe used for further signal processing to compensate for excitationenergy loss along the waveguide. When a waveguide is configured toprovide excitation energy to multiple sample wells, compensation foroverall excitation energy loss along the length of the waveguide may beused to deliver at least a certain amount of excitation energy to eachsample well. Additionally, absorptive elements 9-2104 at the end of awaveguide may be positioned to reduce scattering of any light remainingin the waveguide, as shown in FIG. 9-22.

Additionally or alternatively, monitor sensors may be located atspecific positions along the path of a waveguide. Such monitor sensorsmay be positioned before multi-mode interference splitters, aftermulti-mode interference splitters, before one or more sample wells,and/or after one or more sample wells. Such locations for monitorsensors may be selected based on where excitation energy loss may occurand/or where monitoring of excitation energy loss may be a factor inoverall operation of the integrated device.

In some embodiments, sensors may be located on either side of a pixelarray of the integrated device to monitor input excitation energy andoutput excitation energy from one or more waveguides. The integrateddevice may have one or more grating couplers for coupling the inputexcitation energy into one or more waveguides. The input waveguides maybe split into multiple waveguides, where each split waveguide deliversexcitation energy to a row of sample wells. On one side, the sensors maymonitor position of the input excitation energy on the grating coupler.The sensors on the other side may measure the excitation energy from theend of one or more waveguides after a row of sample wells. In someembodiments, alignment of the excitation source to the integrated devicemay consist of initially centering the excitation energy beam on thegrating coupler using signals from the input sensors indicating thebeam's x-y positioning and further adjusting excitation energy beamparameters, such as focus and/or an incidence angle, from signals froman output sensor at the end of each waveguide. In some instances,signals from the output sensors may be used as a calibration step thatmay be determined for each integrated device prior to using theintegrated device for analysis. Additionally, signals from the outputsensors may be used to normalize measurements obtained with theintegrated device.

FIGS. 9-22 and 9-23 illustrate exemplary arrangements of monitoringsensors where there are two columns of sensors on either side of thepixel array. Each sensor has a center-to-center spacing of twice thepixel pitch in the x and y directions. Such an arrangement of sensorsmay accommodate different configurations of grating couplers. One of thesets of two columns monitor the input excitation energy where a set offour sensors in a square arrangement are used as a quadrant detector, asdiscussed above with reference to FIG. 9-20. A grating coupler 9-2201may be positioned over four sensors 9-2202 in the input columns with alayer opaque to the excitation energy in between the grating coupler andthe sensors. In such a configuration, the fours sensors may be used tomonitor the positioning of the excitation energy beam on the gratingcoupler. For example, the intensity measured by the four sensors may beused for monitoring x-y positioning of the excitation energy beam andalignment onto the grating coupler. The two columns on the output sidemay monitor the excitation energy out of individual waveguides. Asillustrated in FIGS. 9-22, a sensor 9-2204 in the output columns isconnected to an individual waveguide 9-2203. A grating coupler 9-2205may be used to couple the excitation energy from waveguide to the outputsensor. An output sensor may monitor the excitation energy directed intoa waveguide by measuring the power coupled at the end each waveguideafter a row of sample wells. FIG. 9-22 illustrates an exemplaryarrangement of grating couplers for the input waveguides on the inputmonitoring sensors, splitting, by component 9-2206, of the inputwaveguides into waveguides that deliver excitation energy to samplewells 9-2207, and grating couplers at the ends of the waveguidespositioned on output sensors, where each output sensor is used tomonitor a waveguide. FIG. 9-23 illustrates an exemplary arrangement ofan integrated device with two columns of monitoring sensors 9-2307 oneither side of the pixel array to provide an alignment readout of theexcitation energy as discussed above.

D. Multiple Excitation Sources

Multiple excitation sources may provide light having different energiesor wavelengths to the plurality of sample wells. Each of the multipleexcitation sources may provide light having a different characteristicwavelength or energy. One or more markers may be identified based onwhether light from an excitation source excites a marker such that themarker emits a photon. In this manner, markers may be identified basedon their absorption spectra by measuring the response of a sample afterilluminating the sample with light from the different excitationsources. For example, a sample having a marker may be illuminated withlight from a first excitation source followed by light from a secondexcitation source. If the marker emits luminescence in response to beingilluminated by light from the first excitation source, then the markermay have an absorption spectrum that overlaps with the characteristicwavelength of the first excitation source.

In some embodiments, it is possible to use a plurality of excitationsources for the excitation energy. This plurality of sources may be, forexample, implemented as a diode laser bar comprising multiple diodelaser emitters. In the manufacture of laser diodes, multiple emittersare commonly fabricated lithographically on a single substrate, and thendiced into single-emitter pieces for individual packaging. But it isalso possible to dice the substrate into pieces with a plurality ofemitters. In some embodiments, the emitters are nearly identical, andmay be spaced equally from each other to lithographic tolerances,typically of the order of 0.1 micrometer.

To couple the light from multiple emitters onto the integrated device,it is possible to image each emitter onto a separate grating coupler onthe integrated device. FIG. 9-24 shows two ways of coupling theexcitation energy to the integrated device. In both examples shown inFIG. 9-24, the array of emitters is along the dotted line at the left,and the array of grating couplers is along the dotted line at the right.The diagrams illustrate arrays of four emitters and four gratingcouplers, but any number of emitters and couplers is possible.

In the top example of FIG. 9-24A, two lenses are used, with theleft-hand lens being at least as large as the array of emitters, and theright-hand lens being at least as large as the array of gratingcouplers. This example illustrates the case of unity magnification, butany appropriate magnification can be used, as long as each lens is onefocal length from its corresponding array, and as long as the distancebetween the lenses is the sum of their focal lengths.

In the bottom example of FIG. 9-24B, arrays of lenses are used to imagethe emitters to the grating couplers. Each array of lenses can beimplemented as a monolithic or polylithic array of spherical oraspherical lenses, or as a monolithic or polylithic array of cylindricalor acylindrical lenses in the plane of the diagram, supplemented by asingle cylindrical or acylindrical lens in the orthogonal direction.This example illustrates the case of unity magnification, but anyappropriate magnification can be used, as long as the appropriatebeam-expanding or beam-compacting lenses are inserted between the arraysof lenses.

Sample wells and waveguides may be arranged in an integrated device inany suitable way to allow coupling of excitation energy from multipleexcitation sources with a plurality of sample wells. Multiple excitationsources may provide light of different characteristic wavelengths to theplurality of sample wells. A waveguide positioned to receive light fromthe multiple excitation sources may deliver the light to one or moresample wells. An integrated device may include additional components(e.g., power splitter, wavelength combiner) that allow light frommultiple excitation sources to be delivered to multiple sample wells.

Some embodiments relate to an integrated device having a waveguideconfigured to propagate more than one characteristic wavelengths oflight received from one end of the waveguide. Sample wells positionedproximate to the waveguide may couple a portion of the light from thewaveguide. FIG. 9-25A shows an exemplary schematic for coupling lightfrom two excitation sources in an integrated device. A first excitationsource provides light having a first characteristic wavelength, λ1. Asecond excitation source provides light having a second characteristicwavelength, λ2. The integrated device includes two grating couplers: afirst grating coupler configured to couple light from the firstexcitation source having λ1, and a second grating coupler configured tocouple light from the second excitation source having λ2. The integrateddevice includes two power splitters, each coupled to one of the gratingcouplers. The power splitter coupled to the first grating coupler issized and shaped to provide multiple outputs configured to propagatelight having λ1. The power splitter coupled to the second gratingcoupler is sized and shaped to provide multiple outputs configured topropagate light having λ2. A wavelength combiner receives an output fromboth of the power splitters as inputs and is sized and shaped to have anoutput configured to couple both λ1 and λ2 into a waveguide. In thismanner, sample wells positioned proximate to the waveguide may receiveboth λ1 and λ2 from the first and second excitation sources.

Some embodiments relate to an integrated device having a waveguideconfigured to propagate more than one characteristic wavelengths oflight received from opposite ends of the waveguide. Sample wellspositioned proximate to the waveguide may couple a portion of the lightfrom the waveguide. FIG. 9-25B shows an exemplary schematic for couplinglight from two excitation sources in an integrated device. A waveguidein the integrated device are configured to receive light having a firstcharacteristic wavelength, λ1, from one end of the waveguide and lighthaving a second characteristic wavelength, λ2, from another end of thewaveguide. The integrated device includes two grating couplers: a firstgrating coupler configured to couple light from the first excitationsource having λ1, and a second grating coupler configured to couplelight from the second excitation source having λ2. The integrated deviceincludes two power splitters, each coupled to one of the gratingcouplers. The power splitter coupled to the first grating coupler issized and shaped to provide an output configured to propagate lighthaving λ1 and couple with a first end of a waveguide. The power splittercoupled to the second grating coupler is sized and shaped to provide toprovide an output configured to propagate light having λ2 and couplewith a second end of the waveguide.

Some embodiments relate to an integrated device having two waveguidespositioned relative to a sample well such that light propagating in eachof the two waveguides is couple to the sample well. The two waveguidesare coupled to different excitation sources that provide light havingdifferent characteristic wavelengths. FIG. 9-25C shows an exemplaryschematic for coupling light from two excitation sources in anintegrated device. A first waveguide is configured to propagate lighthaving a first characteristic wavelength, λ1. A second waveguide isconfigured to propagate light having a second characteristic wavelength,λ2. A sample well is positioned to couple light having λ1 from the firstwaveguide and light having λ2 from the second waveguide. The firstwaveguide may receive light from an excitation source by a combinationof a grating coupler and/or a power splitter configured to propagatelight having λ1. The second waveguide may receive light from anexcitation source by a combination of a grating coupler and/or powersplitter configured to propagate light having λ2.

V. Example Measurements with the Integrated Device and Excitation Source

Measurements for detecting, analyzing, and/or probing molecules in asample may be obtained using any combination of the integrated device orintegrated device and an excitation source described in the presentapplication. The excitation source may be a pulsed excitation source or,in some instances, a continuous wave source. A luminescent marker taggedto a specific sample may indicate the presence of the sample.Luminescent markers may be distinguished by an excitation energy,luminescence emission wavelength, and/or the lifetime of emission energyemitted by a marker. Markers with similar luminescence emissionwavelength may be identified by determining the lifetime for eachmarker. Additionally, markers with similar lifetimes may be identifiedby the luminescence emission wavelength for each marker. By usingmarkers, where markers are identified by a combination of the temporaland/or spectral properties of the emitted luminescence, a quantitativeanalysis and/or identification of markers and associated samples may beperformed.

Lifetime measurements may be used to determine that a marker is presentin a sample well. The lifetime of a luminescent marker may be identifiedby performing multiple experiments where the luminescent marker isexcited into an excited state and then the time when a photon emits ismeasured. The excitation source is pulsed to produce a pulse ofexcitation energy and directed at the marker. The time between theexcitation pulse and a subsequent photon emission event from aluminescent marker is measured. By repeating such experiments with aplurality of excitation pulses, the number of instances a photon emitswithin a particular time interval may be determined. Such results maypopulate a histogram representing the number of photon emission eventsthat occur within a series of discrete time intervals or time bins. Thenumber of time bins and/or the time interval of each bin may be adjustedto identify a particular set of lifetimes and/or markers.

What follows is a description of example measurements that may be madeto identify luminescent markers in some embodiments. Specifically,examples of distinguishing luminescent markers using only a luminescentlifetime measurement, a joint spectral and luminescent lifetimemeasurement, and only a luminescent lifetime measurement, but using twodifferent excitation energies are discussed. Embodiments are not limitedto the examples detailed below. For example, some embodiments mayidentify the luminescent markers using only spectral measurements.Further details of example measurements may be found in U.S. ProvisionalPatent Application 62/164,482, entitled “METHODS FOR NUCLEIC ACIDSEQUENCING,” filed May 20, 2015, which is incorporated by reference inits entirety.

Any suitable luminescent markers may be used. In some embodiments,commercially available fluorophores may be used. By way of example andnot limitation, the following fluorophores may be used: Atto Rho14(“ATRho14”), Dylight 650 (“D650”), SetaTau 647 (“ST647”), CF 633(“C633”), CF 647 (“C647”), Alexa fluor 647 (“AF647”), BODIPY 630/650(“B630”), CF 640R (“C640R”) and/or Atto 647N (“AT647N”).

Additionally and/or optionally, luminescent markers may be modified inany suitable way to increase the speed and accuracy of the sampleanalysis process. For example, a photostabilizer may be conjugated to aluminescent marker. Examples of photostabilizers include but are notlimited to oxygen scavengers or triplet-state quenchers. Conjugatingphotostabilizers to the luminescent marker may increase the rate ofphotons emitted and may also reduce a “blinking” effect where theluminescent marker does not emit photons. In some embodiments, when abiological event occurs on the millisecond scale, an increased rate ofphoton emission may increase the probability of detection of thebiological event. Increased rates of photon events may subsequentlyincrease the signal-to-noise ratio of luminescence signal and increasethe rate at which lifetime measurements are made, leading to a fasterand more accurate sample analysis.

Furthermore, the environment in a sample well of an integrated devicemay be tuned to engineer the lifetime of the markers as needed. This canbe achieved by recognizing that the lifetime of a marker is effected bythe density of state of the marker, which can be tuned using theenvironment. For example, the farther a marker is from the bottom metallayer of the sample well, the longer the lifetime. Accordingly, toincrease the lifetime of a marker, the depth of a bottom surface of asample well, such as a divot, may extend a certain distance from a metallayer. Also, the materials used to form the sample well can affect thelifetime of the markers. While different markers typically have theirlifetimes shifted in the same direction (e.g., either longer orshorter), the affect may scale differently for different markers.Accordingly, two markers that cannot be distinguished via lifetimemeasurements in free-space may be engineered to be distinguishable byfabricating the sample well environment to adjust the lifetimes of thevarious markers.

A. Single Molecule Detection and Sequencing

According to an aspect of the present application, a single molecule canbe identified (e.g., distinguished from other possible molecules in areaction sample) based on one or more properties of a series of photonsthat are emitted from the molecule when it is exposed to a plurality ofseparate light pulses. In some embodiments, the molecule is labelledwith a luminescent marker. In some embodiments, the luminescent markeris a fluorophore. In some embodiments, the luminescent marker can beidentified or distinguished based on a property of the luminescentmarker. Properties of a luminescent marker (e.g., a fluorophore)include, but are not limited to luminescent lifetimes, absorptionspectra, emission spectra, luminescence quantum yield, and luminescentintensity, and combinations of two or more thereof.

A biological sample may be processed in preparation for detection (e.g.,sequencing). Such processing can include isolation and/or purificationof the biomolecule (e.g., nucleic acid molecule) from the biologicalsample, and generation of more copies of the biomolecule. In someexamples, one or more nucleic acid molecules are isolated and purifiedform a bodily fluid or tissue of the subject, and amplified throughnucleic acid amplification, such as polymerase chain reaction (PCR).Then, the one or more nucleic acids molecules or subunits thereof can beidentified, such as through sequencing. However, in some embodimentsnucleic acid samples can be evaluated (e.g., sequenced) as described inthis application without requiring amplification.

Sequencing can include the determination of individual subunits of atemplate biomolecule (e.g., nucleic acid molecule) by synthesizinganother biomolecule that is complementary or analogous to the template,such as by synthesizing a nucleic acid molecule that is complementary toa template nucleic acid molecule and identifying the incorporation ofnucleotides with time (e.g., sequencing by synthesis). As analternative, sequencing can include the direct identification ofindividual subunits of the biomolecule.

During sequencing, a polymerizing enzyme may couple (e.g., attach) to apriming location of a target nucleic acid molecule. The priming locationcan be a primer that is complementary to the target nucleic acidmolecule. As an alternative the priming location is a gap or nick thatis provided within a double stranded segment of the target nucleic acidmolecule. A gap or nick can be from 0 to at least 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 20, 30, or 40 nucleotides in length. A nick can provide abreak in one strand of a double stranded sequence, which can provide apriming location for a polymerizing enzyme, such as, for example, astrand displacing polymerase enzyme.

In some cases, a sequencing primer can be annealed to a target nucleicacid molecule that may or may not be immobilized to a solid support,such as a sample well. In some embodiments, a sequencing primer may beimmobilized to a solid support and hybridization of the target nucleicacid molecule also immobilizes the target nucleic acid molecule to thesolid support. Via the action of an enzyme (e.g., a polymerase) capableof adding or incorporating a nucleotide to the primer, nucleotides canbe added to the primer in 5′ to 3′, template bound fashion. Suchincorporation of nucleotides to a primer (e.g., via the action of apolymerase) can generally be referred to as a primer extension reaction.Each nucleotide can be associated with a detectable marker as part of atag that can be detected and used to determine each nucleotideincorporated into the primer and, thus, a sequence of the newlysynthesized nucleic acid molecule. Via sequence complementarity of thenewly synthesized nucleic acid molecule, the sequence of the targetnucleic acid molecule can also be determined. In some cases, annealingof a sequencing primer to a target nucleic acid molecule andincorporation of nucleotides to the sequencing primer can occur atsimilar reaction conditions (e.g., the same or similar reactiontemperature) or at differing reaction conditions (e.g., differentreaction temperatures). Moreover, some sequencing by synthesis methodscan include the presence of a population of target nucleic acidmolecules (e.g, copies of a target nucleic acid) and/or a step ofamplification of the target nucleic acid to achieve a population oftarget nucleic acids.

The single-stranded target nucleic acid template can be contacted with asequencing primer, dNTPs, polymerase and other reagents necessary fornucleic acid synthesis. In some embodiments, all appropriate dNTPs canbe contacted with the single-stranded target nucleic acid templatesimultaneously (e.g., all dNTPs are simultaneously present) such thatincorporation of dNTPs can occur continuously. In other embodiments, thedNTPs can be contacted with the single-stranded target nucleic acidtemplate sequentially, where the single-stranded target nucleic acidtemplate is contacted with each appropriate dNTP separately, withwashing steps in between contact of the single-stranded target nucleicacid template with differing dNTPs. Such a cycle of contacting thesingle-stranded target nucleic acid template with each dNTP separatelyfollowed by washing can be repeated for each successive base position ofthe single-stranded target nucleic acid template to be identified.

The sequencing primer anneals to the single-stranded target nucleic acidtemplate and the polymerase consecutively incorporates the dNTPs (orother deoxyribonucleoside polyphosphate) to the primer via thesingle-stranded target nucleic acid template. The unique luminescentmarker associated with each incorporated dNTP can be excited with theappropriate excitation light during or after incorporation of the dNTPto the primer and its emission can be subsequently detected, using, anysuitable device(s) and/or method(s), including devices and methods fordetection described elsewhere herein. Detection of a particular emissionof light can be attributed to a particular dNTP incorporated. Thesequence obtained from the collection of detected luminescent markerscan then be used to determine the sequence of the single-stranded targetnucleic acid template via sequence complementarity.

While the present disclosure makes reference to dNTPs, devices, systemsand methods provided herein may be used with various types ofnucleotides, such as ribonucleotides and deoxyribonucleotides (e.g.,deoxyribonucleoside polyphophates with at least 4, 5, 6, 7, 8, 9, or 10phosphate groups). Such ribonucleotides and deoxyribonucleotides canform various types of tags by using a linker to attach a marker to aribonucleotide or a deoxyribonucleotide.

Signals emitted upon the incorporation of nucleosides can be stored inmemory and processed at a later point in time to determine the sequenceof the target nucleic acid template. This may include comparing thesignals to a reference signals to determine the identities of theincorporated nucleosides as a function of time. Alternative or inaddition to, signal emitted upon the incorporation of nucleoside can becollected and processed in real time (i.e., upon nucleosideincorporation) to determine the sequence of the target nucleic acidtemplate in real time.

Nucleic acid sequencing of a plurality of single-stranded target nucleicacid templates may be completed where multiple sample wells areavailable, as is the case in devices described elsewhere herein. Eachsample well can be provided with a single-stranded target nucleic acidtemplate and a sequencing reaction can be completed in each sample well.Each of the sample wells may be contacted with the appropriate reagents(e.g., dNTPs, sequencing primers, polymerase, co-factors, appropriatebuffers, etc.) necessary for nucleic acid synthesis during a primerextension reaction and the sequencing reaction can proceed in eachsample well. In some embodiments, the multiple sample wells arecontacted with all appropriate dNTPs simultaneously. In otherembodiments, the multiple sample wells are contacted with eachappropriate dNTP separately and each washed in between contact withdifferent dNTPs. Incorporated dNTPs can be detected in each sample welland a sequence determined for the single-stranded target nucleic acid ineach sample well as is described above.

Embodiments directed toward single molecule RNA sequencing may use anyreverse transcriptase that is capable of synthesizing complementary DNA(cDNA) from an RNA template. In such embodiments, a reversetranscriptase can function in a manner similar to polymerase in thatcDNA can be synthesized from an RNA template via the incorporation ofdNTPs to a reverse transcription primer annealed to an RNA template. ThecDNA can then participate in a sequencing reaction and its sequencedetermined as described above. The determined sequence of the cDNA canthen be used, via sequence complementarity, to determine the sequence ofthe original RNA template. Examples of reverse transcriptases includeMoloney Murine Leukemia Virus reverse transcriptase (M-MLV), avianmyeloblastosis virus (AMV) reverse transcriptase, human immunodeficiencyvirus reverse transcriptase (HIV-1) and telomerase reversetranscriptase.

Sequence reads can be used to reconstruct a longer region of a genome ofa subject (e.g., by alignment). Reads can be used to reconstructchromosomal regions, whole chromosomes, or the whole genome. Sequencereads or a larger sequence generated from such reads can be used toanalyze a genome of a subject, such as to identify variants orpolymorphisms. Examples of variants include, but are not limited to,single nucleotide polymorphisms (SNPs) including tandem SNPs,small-scale multi-base deletions or insertions, also referred to asindels or deletion insertion polymorphisms (DIPs), Multi-NucleotidePolymorphisms (MNPs), Short Tandem Repeats (STRs), deletions, includingmicrodeletions, insertions, including microinsertions, structuralvariations, including duplications, inversions, translocations,multiplications, complex multi-site variants, copy number variations(CNV). Genomic sequences can comprise combinations of variants. Forexample, genomic sequences can encompass the combination of one or moreSNPs and one or more CNVs.

In some embodiments, the molecules are identified or distinguished basedon luminescent lifetime. In some embodiments, the molecules areidentified or distinguished based on luminescent intensity. In someembodiments, the molecules are identified or distinguished based on thewavelength of the delivered excitation energy necessary to observe anemitted photon. In some embodiments, the molecules are identified ordistinguished based on the wavelength of an emitted photon. In someembodiments, the molecules are identified or distinguished based on bothluminescent lifetime and the wavelength of the delivered excitationenergy necessary to observe an emitted photon. In some embodiments, themolecules are identified or distinguished based on both a luminescentintensity and the wavelength of the delivered excitation energynecessary to observe an emitted photon. In some embodiments, themolecules are identified or distinguished based on luminescent lifetime,luminescent intensity, and the wavelength of the delivered excitationenergy necessary to observe an emitted photon. In some embodiments, themolecules are identified or distinguished based on both luminescentlifetime and the wavelength of an emitted photon. In some embodiments,the molecules are identified or distinguished based on both aluminescent intensity and the wavelength of an emitted photon. In someembodiments, the molecules are identified or distinguished based onluminescent lifetime, luminescent intensity, and the wavelength of anemitted photon.

In certain embodiments, different types of molecules in a reactionmixture or experiment are labeled with different luminescent markers. Insome embodiments, the different markers have different luminescentproperties which can be distinguished. In some embodiments, thedifferent markers are distinguished by having different luminescentlifetimes, different luminescent intensities, different wavelengths ofemitted photons, or a combination thereof. The presence of a pluralityof types of molecules with different luminescent markers may allow fordifferent steps of a complex reaction to be monitored, or for differentcomponents of a complex reaction product to be identified. In someembodiments, the order in which the different types of molecules reactor interact can be determined.

In certain embodiments, the luminescent properties of a plurality oftypes of molecules with different luminescent markers are used toidentify the sequence of a biomolecule, such as a nucleic acid orprotein. In some embodiments, the luminescent properties of a pluralityof types of molecules with different luminescent markers are used toidentify single molecules as they are incorporated during the synthesisof a biomolecule. In some embodiments, the luminescent properties of aplurality of types of nucleotides with different luminescent markers areused to identify single nucleotides as they are incorporated during asequencing reaction. In some embodiments, methods, compositions, anddevices described in the application can be used to identify a series ofnucleotides that are incorporated into a template-dependent nucleic acidsequencing reaction product synthesized by a polymerase enzyme.

In certain embodiments, the template-dependent nucleic acid sequencingproduct is carried out by naturally occurring nucleic acid polymerases.In some embodiments, the polymerase is a mutant or modified variant of anaturally occurring polymerase. In some embodiments, thetemplate-dependent nucleic acid sequence product will comprise one ormore nucleotide segments complementary to the template nucleic acidstrand. In one aspect, the application provides a method of determiningthe sequence of a template (or target) nucleic acid strand bydetermining the sequence of its complementary nucleic acid strand.

In another aspect, the application provides methods of sequencing targetnucleic acids by sequencing a plurality of nucleic acid fragments,wherein the target nucleic acid comprises the fragments. In certainembodiments, the method comprises combining a plurality of fragmentsequences to provide a sequence or partial sequence for the parenttarget nucleic acid. In some embodiments, the step of combining isperformed by computer hardware and software. The methods describedherein may allow for a set of related target nucleic acids, such as anentire chromosome or genome to be sequenced.

The term “genome” generally refers to an entirety of an organism'shereditary information. A genome can be encoded either in DNA or in RNA.A genome can comprise coding regions that code for proteins as well asnon-coding regions. A genome can include the sequence of all chromosomestogether in an organism. For example, the human genome has a total of 46chromosomes. The sequence of all of these together constitutes the humangenome. In some embodiments, the sequence of an entire genome isdetermined. However, in some embodiments, sequence information for asubset of a genome (e.g., one or a few chromosomes, or regions thereof)or for one or a few genes (or fragments thereof) are sufficient fordiagnostic, prognostic, and/or therapeutic applications.

In some embodiments, the target nucleic acid molecule used in singlemolecule sequencing is a single-stranded target nucleic acid (e.g.deoxyribonucleic acid (DNA), DNA derivatives, ribonucleic acid (RNA),RNA derivatives) template that is added or immobilized to a sample wellcontaining at least one additional component of a sequencing reaction(e.g., a polymerase such as, a DNA polymerase, a sequencing primer)immobilized or attached to a solid support such as the bottom of thesample well. In some cases, a sequencing primer can be annealed to atarget nucleic acid molecule that may or may not be immobilized to asolid support, such as a sample well (e.g., nanoaperture). In someembodiments, a sequencing primer may be immobilized to a solid supportand hybridization of the target nucleic acid molecule also immobilizesthe target nucleic acid molecule to the solid support. In someembodiments, a polymerase is immobilized to a solid support and solubleprimer and target nucleic acid are contacted to the polymerase. However,in some embodiments a complex comprising a polymerase, a target nucleicacid and a primer is formed in solution and the complex is immobilizedto a solid support (e.g., via immobilization of the polymerase, primer,and/or target nucleic acid).

Under appropriate conditions, a polymerase enzyme that is contacted toan annealed primer/target nucleic acid can add or incorporate one ormore nucleotides onto the primer, and nucleotides can be added to theprimer in a 5′ to 3′, template bound fashion. Such incorporation ofnucleotides onto a primer (e.g., via the action of a polymerase) cangenerally be referred to as a primer extension reaction. Each nucleotidecan be associated with a detectable marker that can be detected andidentified (e.g., based on its luminescent lifetime, emission spectra,absorption spectra, and/or other characteristics) and used to determineeach nucleotide incorporated into the primer and, thus, a sequence ofthe newly synthesized nucleic acid molecule. Via sequencecomplementarity of the newly synthesized nucleic acid molecule, thesequence of the target nucleic acid molecule can also be determined. Insome embodiments, sequencing by synthesis methods can include thepresence of a population of target nucleic acid molecules (e.g., copiesof a target nucleic acid) and/or a step of amplification of the targetnucleic acid to achieve a population of target nucleic acids. However,in some embodiments sequencing by synthesis is used to determine thesequence of a single molecule in each reaction that is being evaluated(and nucleic acid amplification is not required to prepare the targettemplate for sequencing). In some embodiments, a plurality of singlemolecule sequencing reactions are performed in parallel (e.g., on asingle integrated device or chip) according to aspects of the presentapplication.

Embodiments are capable of sequencing single nucleic acid molecules withhigh accuracy and long read lengths, such as an accuracy of at leastabout 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%,99.99%, 99.999%, or 99.9999%, and/or read lengths greater than or equalto about 10 base pairs (bp), 50 bp, 100 bp, 200 bp, 300 bp, 400 bp, 500bp, 1000 bp, 10,000 bp, 20,000 bp, 30,000 bp, 40,000 bp, 50,000 bp, or100,000 bp.

The target nucleic acid molecule or the polymerase can be attached to asample wall, such as at the bottom of the sample well directly orthrough a linker. The sample well (e.g., nanoaperture) also can containany other reagents needed for nucleic acid synthesis via a primerextension reaction, such as, for example suitable buffers, co-factors,enzymes (e.g., a polymerase) and deoxyribonucleoside polyphosphates,such as, e.g., deoxyribonucleoside triphosphates, includingdeoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP),deoxyguanosine triphosphate (dGTP), deoxyuridine triphosphate (dUTP) anddeoxythymidine triphosphate (dTTP) dNTPs, that include luminescentmarkers, such as fluorophores. In some embodiments, each class of dNTPs(e.g. adenine-containing dNTPs (e.g., dATP), cytosine-containing dNTPs(e.g., dCTP), guanine-containing dNTPs (e.g., dGTP), uracil-containingdNTPs (e.g., dUTPs) and thymine-containing dNTPs (e.g., dTTP)) isconjugated to a distinct luminescent marker such that detection of lightemitted from the marker indicates the identity of the dNTP that wasincorporated into the newly synthesized nucleic acid. Emitted light fromthe luminescent marker can be detected and attributed to its appropriateluminescent marker (and, thus, associated dNTP) via any suitable deviceand/or method, including such devices and methods for detectiondescribed elsewhere herein. The luminescent marker may be conjugated tothe dNTP at any position such that the presence of the luminescentmarker does not inhibit the incorporation of the dNTP into the newlysynthesized nucleic acid strand or the activity of the polymerase. Insome embodiments, the luminescent marker is conjugated to the terminalphosphate (the gamma phosphate) of the dNTP.

In some embodiments, the sequencing primer anneals to thesingle-stranded target nucleic acid template and the polymeraseconsecutively incorporates the dNTPs (or other deoxyribonucleosidepolyphosphate) to the primer via the single-stranded target nucleic acidtemplate. The unique luminescent marker associated with eachincorporated dNTP can be excited with the appropriate excitation lightduring or after incorporation of the dNTP to the primer and its emissioncan be subsequently detected, using, any suitable device(s) and/ormethod(s), including devices and methods for detection describedelsewhere herein. Detection of a particular emission of light (e.g.,having a particular emission lifetime, intensity, and/or combinationthereof) can be attributed to a particular dNTP incorporated. Thesequence obtained from the collection of detected luminescent markerscan then be used to determine the sequence of the single-stranded targetnucleic acid template via sequence complementarity.

As an example, FIG. 10-1 schematically illustrates the setup of a singlemolecule nucleic acid sequencing method. The example is not meant tolimit the invention in any way. 610 is a sample well (e.g.,nanoaperture) configured to contain a single complex comprising anucleic acid polymerase 601, a target nucleic acid 602 to be sequenced,and a primer 604. In this example, a bottom region of sample well 610 isdepicted as a target volume 620. In FIG. 10-1 the complex comprisingpolymerase 601 is confined in target volume 620. The complex mayoptionally be immobilized by attachment to a surface of the sample well.In this example the complex is immobilized by a linker 603 comprisingone or more biomolecules (e.g., biotin) suitable for attaching thelinker to the polymerase 601.

The volume of the sample well also contains a reaction mixture withsuitable solvent, buffers, and other additives necessary for thepolymerase complex to synthesize a nucleic acid strand. The reactionmixture also contains a plurality of types of luminescently labelednucleotides. Each type of nucleotide is represented by the symbols *-A,@-T, $-G, #-C, wherein A, T, G, and C represent the nucleotide base, andthe symbols *, @, $, and # represent a unique luminescent markerattached to each nucleotide, through linker -. In FIG. 10-1, a #-Cnucleotide is currently being incorporated into the complementary strand602. The incorporated nucleotide is located within the target volume620.

FIG. 10-1 also indicates with arrows the concept of an excitation energybeing delivered to a vicinity of the target volume, and luminescencebeing emitted towards a detector. The arrows are schematic, and are notmeant to indicate the particular orientation of excitation energydelivery or luminescence. Some luminescences may emit on a vector whichis not directed to the detector (e.g., towards the sidewall of thesample well) and may not be detected.

FIG. 10-2 schematically illustrates a sequencing process in a singlesample well over time. Stages A through D depict a sample well with apolymerase complex as in FIG. 10-1. Stage A depicts the initial statebefore any nucleotides have been added to the primer. Stage B depictsthe incorporation event of a luminescently labeled nucleotide (#-C).Stage C, depicts the period between incorporation events. In thisexample, nucleotide C has been added to the primer, and the label andlinker previously attached to the luminescently labeled nucleotide (#-C)has been cleaved. Stage D depicts a second incorporation event of aluminescently labeled nucleotide (*-A). The complementary strand afterStage D consists of the primer, a C nucleotide, and an A nucleotide.

Stage A and C, both depict the periods before or between incorporationevents, which are indicated in this example to last for about 10milliseconds. In stages A and C, because there is no nucleotide beingincorporated, there is no luminescently labeled nucleotide in the targetvolume (not drawn in FIG. 10-2), though background luminescence orspurious luminescence from a luminescently labeled nucleotide which isnot being incorporated may be detected. Stage B and D show incorporationevents of different nucleotides (#-C, and *-A, respectively). In thisexample these events are also indicated to last for about 10milliseconds.

The row labeled “Raw bin data” depicts the data generated during eachStage. Throughout the example experiment, a plurality of pulses of lightis delivered to the vicinity of the target volume. For each pulse adetector is configured to record any emitted photon received by thedetector, and assign the detected photon to a time bin based on the timeduration since the last pulse of excitation energy. In this examplethere are 3 bins, and the “Raw bin data” records a value of 1 (shortestbars), 2 (medium bars), or 3 (longest bars), corresponding to theshortest, middle, and longest bins, respectively. Each bar indicatesdetection of an emitted photon.

Since there is no luminescently labeled nucleotide present in the targetvolume for Stage A or C, there are no photons detected. For each ofStage B and D a plurality of luminescences is detected during theincorporation event. Luminescent marker # has a shorter luminescencelifetime than luminescent marker *. The Stage B data is thus depicted ashaving recorded lower average bin values, than Stage D where the binvalues are higher.

The row labeled “Processed data” depicts raw data which has beenprocessed to indicate the number (counts) of emitted photons at timesrelative to each pulse. In this example the data is only processed todetermine luminescent lifetime, but the data may also be evaluated forother luminescent properties, such as luminescent intensity or thewavelength of the absorbed or emitted photons. The exemplary processeddata approximates an exponential decay curve characteristic for theluminescence lifetime of the luminescent marker in the target volume.Because luminescent marker # has a shorter luminescence lifetime thanluminescent marker *, the processed data for Stage B has fewer counts atlonger time durations, while the processed data for Stage D hasrelatively more counts at longer time durations.

The example experiment of FIG. 10-2 would identify the first twonucleotides added to the complementary strand as CA. For DNA, thesequence of the target strand immediately after the region annealed tothe primer would thus be identified as GT. In this example thenucleotides C and A could be distinguished from amongst the plurality ofC, G, T, and A, based on luminescent lifetime alone. In someembodiments, other properties, such as the luminescent intensity or thewavelength of the absorbed or emitted photons may be necessary todistinguish one or more particular nucleotides.

B. Luminescent Properties

As described herein, a luminescent molecule is a molecule that absorbsone or more photons and may subsequently emit one or more photons afterone or more time durations. The luminescence of the molecule isdescribed by several parameters, including but not limited toluminescent lifetime, absorption and/or emission spectra, luminescentquantum yield, and luminescent intensity.

The emitted photon from a luminescent emission event will emit at awavelength within a spectral range of possible wavelengths. Typicallythe emitted photon has a longer wavelength (e.g., has less energy or isred-shifted) compared to the wavelength of the excitation photon. Incertain embodiments, a molecule is identified my measuring thewavelength of an emitted photon. In certain embodiments, a molecule isidentified by measuring the wavelength of a plurality of emittedphotons. In certain embodiments, a molecule is identified by measuringthe emission spectrum.

Luminescent lifetime refers to a time duration (e.g., emission decaytime) associated with an excitation event and an emission event. In someembodiments, luminescent lifetime is expressed as the constant in anequation of exponential decay. In some embodiments, wherein there areone or more pulse events delivering excitation energy, the time durationis the time between the pulse and the subsequent emission event.

Luminescent quantum yield refers to the fraction of excitation events ata given wavelength or within a given spectral range that lead to anemission event, and is typically less than 1. In some embodiments, theluminescent quantum yield of a molecule described herein is between 0and about 0.001, between about 0.001 and about 0.01, between about 0.01and about 0.1, between about 0.1 and about 0.5, between about 0.5 and0.9, or between about 0.9 and 1. In some embodiments, a molecule isidentified by determining or estimating the luminescent quantum yield.

As used herein for single molecules luminescent intensity refers to thenumber of emitted photons per unit time that are emitted by a moleculewhich is being excited by delivery of a pulsed excitation energy. Insome embodiments, the luminescent intensity refers to the detectednumber of emitted photons per unit time that are emitted by a moleculewhich is being excited by delivery of a pulsed excitation energy, andare detected by a particular sensor or set of sensors.

In one aspect, the application provides a method of determining theluminescent lifetime of a single luminescent molecule comprising:providing the luminescent molecule in a target volume; delivering aplurality of pulses of an excitation energy to a vicinity of the targetvolume; and detecting a plurality of luminescences from the luminescentmolecule. In some embodiments, the method further comprises recording aplurality of time durations between each pair of pulses andluminescences and evaluating the distribution of the plurality of timedurations between each pair of pulses and luminescences.

C. Luminescently Labeled Nucleotides

In one aspect, methods and compositions described herein comprises oneor more luminescently labeled nucleotide. In certain embodiments, one ormore nucleotides comprise deoxyribose nucleosides. In some embodiments,all nucleotides comprises deoxyribose nucleosides. In certainembodiments, one or more nucleotides comprise ribose nucleosides. Insome embodiments, all nucleotides comprise ribose nucleosides. In someembodiments, one or more nucleotides comprise a modified ribose sugar orribose analog (e.g., a locked nucleic acid). In some embodiments, one ormore nucleotides comprise naturally occurring bases (e.g., cytosine,guanine, adenine, thymine, uracil). In some embodiments, one or morenucleotides comprise derivatives or analogs of cytosine, guanine,adenine, thymine, or uracil.

In certain embodiments, a method comprises the step of exposing apolymerase complex to a plurality of luminescently labeled nucleotides.In certain embodiments, a composition or device comprises a reactionmixture comprising a plurality of luminescently labeled nucleotides. Insome embodiments, the plurality of nucleotides comprises four types ofnucleotides. In some embodiments, the four types of nucleotides eachcomprise one of cytosine, guanine, adenine, and thymine. In someembodiments, the four types of nucleotides each comprise one ofcytosine, guanine, adenine, and uracil.

The term “nucleic acid,” as used herein, generally refers to a moleculecomprising one or more nucleic acid subunits. A nucleic acid may includeone or more subunits selected from adenosine (A), cytosine (C), guanine(G), thymine (T) and uracil (U), or variants thereof. In some examples,a nucleic acid is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA),or derivatives thereof. A nucleic acid may be single-stranded or doublestranded. A nucleic acid may be circular.

The term “nucleotide,” as used herein, generally refers to a nucleicacid subunit, which can include A, C, G, T or U, or variants or analogsthereof. A nucleotide can include any subunit that can be incorporatedinto a growing nucleic acid strand. Such subunit can be an A, C, G, T,or U, or any other subunit that is specific to one or more complementaryA, C, G, T or U, or complementary to a purine (i.e., A or G, or variantor analogs thereof) or a pyrimidine (i.e., C, T or U, or variant oranalogs thereof). A subunit can enable individual nucleic acid bases orgroups of bases (e.g., AA, TA, AT, GC, CG, CT, TC, GT, TG, AC, CA, oruracil-counterparts thereof) to be resolved.

A nucleotide generally includes a nucleoside and at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, or more phosphate (PO3) groups. A nucleotide can includea nucleobase, a five-carbon sugar (either ribose or deoxyribose), andone or more phosphate groups. Ribonucleotides are nucleotides in whichthe sugar is ribose. Deoxyribonucleotides are nucleotides in which thesugar is deoxyribose. A nucleotide can be a nucleoside monophosphate ora nucleoside polyphosphate. A nucleotide can be a deoxyribonucleosidepolyphosphate, such as, e.g., a deoxyribonucleoside triphosphate, whichcan be selected from deoxyadenosine triphosphate (dATP), deoxycytidinetriphosphate (dCTP), deoxyguanosine triphosphate (dGTP), deoxyuridinetriphosphate (dUTP) and deoxythymidine triphosphate (dTTP) dNTPs, thatinclude detectable markers (e.g., fluorophores).

A nucleoside polyphosphate can have ‘n’ phosphate groups, where ‘n’ is anumber that is greater than or equal to 2, 3, 4, 5, 6, 7, 8, 9, or 10.Examples of nucleoside polyphosphates include nucleoside diphosphate andnucleoside triphosphate. A nucleotide can be a terminal phosphatelabeled nucleoside, such as a terminal phosphate labeled nucleosidepolyphosphate. Such marker can be a luminescent (e.g., fluorescent orchemiluminescent) marker, a fluorogenic marker, a colored marker, achromogenic marker, a mass tag, an electrostatic marker, or anelectrochemical marker. A marker can be coupled to a terminal phosphatethrough a linker. The linker can include, for example, at least one or aplurality of hydroxyl groups, sulfhydryl groups, amino groups orhaloalkyl groups, which may be suitable for forming, for example, aphosphate ester, a thioester, a phosphoramidate or an alkyl phosphonatelinkage at the terminal phosphate of a natural or modified nucleotide. Alinker can be cleavable so as to separate a marker from the terminalphosphate, such as with the aid of a polymerization enzyme. Examples ofnucleotides and linkers are provided in U.S. Pat. No. 7,041,812, whichis entirely incorporated herein by reference.

D. Markers

In certain embodiments, the incorporated molecule is a luminescentmolecule, e.g., without attachment of a distinct luminescent marker.Typical nucleotide and amino acids are not luminescent, or do notluminesce within suitable ranges of excitation and emission energies. Incertain embodiments, the incorporated molecule comprises a luminescentmarker. In certain embodiments, the incorporated molecule is aluminescently labeled nucleotide. In certain embodiments, theincorporated molecule is a luminescently labeled amino acid orluminescently labeled tRNA. In some embodiments, a luminescently labelednucleotide comprises a nucleotide and a luminescent marker. In someembodiments, a luminescently labeled nucleotide comprises a nucleotide,a luminescent marker, and a linker. In some embodiments, the luminescentmarker is a fluorophore.

For nucleotide sequencing, certain combinations of luminescently labelednucleotides may be preferred. In some embodiments, at least one of theluminescently labeled nucleotides comprises a cyanine dye, or analogthereof. In some embodiments, at least one luminescently labelednucleotides comprises a rhodamine dye, or analog thereof. In someembodiments, at least one of the luminescently labeled nucleotidescomprises a cyanine dye, or analog thereof, and at least oneluminescently labeled nucleotides comprises a rhodamine dye, or analogthereof.

In certain embodiments, the luminescent marker is a dye selected fromTable FL-1. The dyes listed in Table FL-1 are non-limiting, and theluminescent markers of the application may include dyes not listed inTable FL-1. In certain embodiments, the luminescent markers of one ormore luminescently labeled nucleotides is selected from Table FL-1. Incertain embodiments, the luminescent markers of four or moreluminescently labeled nucleotides is selected from Table FL-1.

TABLE FL-1 Exemplary fluorophores. Fluorophores 5/6-CarboxyrhodamineCF532 DyLight 747-B3 Dyomics-781 6G 5-Carboxyrhodamine CF543 DyLight747-B4 Dyomics-782 6G 6-Carboxyrhodamine CF546 DyLight 755 Dyomics-8006G 6-TAMRA CF555 DyLight 766Q Dyomics-831 Abberior Star 440SXP CF568DyLight 775-B2 eFluor 450 Abberior Star 470SXP CF594 DyLight 775-B3Eosin Abberior Star 488 CF620R DyLight 775-B4 FITC Abberior Star 512CF633 DyLight 780-B1 Fluorescein Abberior Star 520SXP CF633-V1 DyLight780-B2 HiLyte Fluor 405 Abberior Star 580 CF640R DyLight 780-B3 HiLyteFluor 488 Abberior Star 600 CF640R-V1 DyLight 800 HiLyte Fluor 532Abberior Star 635 CF640R-V2 DyLight 830-B2 HiLyte Fluor 555 AbberiorStar 635P CF660C Dyomics-350 HiLyte Fluor 594 Abberior Star RED CF660RDyomics-350XL HiLyte Fluor 647 Alexa Fluor 350 CF680 Dyomics-360XLHiLyte Fluor 680 Alexa Fluor 405 CF680R Dyomics-370XL HiLyte Fluor 750Alexa Fluor 430 CF680R-V1 Dyomics-375XL IRDye 680LT Alexa Fluor 480CF750 Dyomics-380XL IRDye 750 Alexa Fluor 488 CF770 Dyomics-390XL IRDye800CW Alexa Fluor 514 CF790 Dyomics-405 JOE Alexa Fluor 532 Chromeo 642Dyomics-415 LightCycler 640R Alexa Fluor 546 Chromis 425N Dyomics-430LightCycler Red 610 Alexa Fluor 555 Chromis 500N Dyomics-431 LightCyclerRed 640 Alexa Fluor 568 Chromis 515N Dyomics-478 LightCycler Red 670Alexa Fluor 594 Chromis 530N Dyomics-480XL LightCycler Red 705 AlexaFluor 610-X Chromis 550A Dyomics-481XL Lissamine Rhodamine B Alexa Fluor633 Chromis 550C Dyomics-485XL Napthofluorescein Alexa Fluor 647 Chromis550Z Dyomics-490 Oregon Green 488 Alexa Fluor 660 Chromis 560NDyomics-495 Oregon Green 514 Alexa Fluor 680 Chromis 570N Dyomics-505Pacific Blue Alexa Fluor 700 Chromis 577N Dyomics-510XL Pacific GreenAlexa Fluor 750 Chromis 600N Dyomics-511XL Pacific Orange Alexa Fluor790 Chromis 630N Dyomics-520XL PET AMCA Chromis 645A Dyomics-521XL PF350ATTO 390 Chromis 645C Dyomics-530 PF405 ATTO 425 Chromis 645ZDyomics-547 PF415 ATTO 465 Chromis 678A Dyomics-547P1 PF488 ATTO 488Chromis 678C Dyomics-548 PF505 ATTO 495 Chromis 678Z Dyomics-549 PF532ATTO 514 Chromis 770A Dyomics-549P1 PF546 ATTO 520 Chromis 770CDyomics-550 PF555P ATTO 532 Chromis 800A Dyomics-554 PF568 ATTO 542Chromis 800C Dyomics-555 PF594 ATTO 550 Chromis 830A Dyomics-556 PF610ATTO 565 Chromis 830C Dyomics-560 PF633P ATTO 590 Cy3 Dyomics-590 PF647PATTO 610 Cy3.5 Dyomics-591 Quasar 570 ATTO 620 Cy3B Dyomics-594 Quasar670 ATTO 633 Cy5 Dyomics-601XL Quasar 705 ATTO 647 DyLight 350Dyomics-605 Rhoadmine 123 ATTO 647N DyLight 405 Dyomics-610 Rhodamine 6GATTO 655 DyLight 415-Co1 Dyomics-615 Rhodamine B ATTO 665 DyLight 425QDyomics-630 Rhodamine Green ATTO 680 DyLight 485-LS Dyomics-631Rhodamine Green-X ATTO 700 DyLight 488 Dyomics-632 Rhodamine Red ATTO725 DyLight 504Q Dyomics-633 ROX ATTO 740 DyLight 510-LS Dyomics-634Seta 375 ATTO Oxa12 DyLight 515-LS Dyomics-635 Seta 470 ATTO Rho101DyLight 521-LS Dyomics-636 Seta 555 ATTO Rho11 DyLight 530-R2Dyomics-647 Seta 632 ATTO Rho12 DyLight 543Q Dyomics-647P1 Seta 633 ATTORho13 DyLight 550 Dyomics-648 Seta 650 ATTO Rho14 DyLight 554-R0Dyomics-648P1 Seta 660 ATTO Rho36 DyLight 554-R1 Dyomics-649 Seta 670ATTO Rho6G DyLight 590-R2 Dyomics-649P1 Seta 680 ATTO Thio12 DyLight 594Dyomics-650 Seta 700 BD Horizon V450 DyLight 610-B1 Dyomics-651 Seta 750BODIPY 493/501 DyLight 615-B2 Dyomics-652 Seta 780 BODIPY 530/550DyLight 633 Dyomics-654 Seta APC-780 BODIPY 558/568 DyLight 633-B1Dyomics-675 Seta PerCP-680 BODIPY 564/570 DyLight 633-B2 Dyomics-676Seta R-PE-670 BODIPY 576/589 DyLight 650 Dyomics-677 Seta646 BODIPY581/591 DyLight 655-B1 Dyomics-678 SeTau 380 BODIPY 630/650 DyLight655-B2 Dyomics-679P1 SeTau 405 BODIPY 650/665 DyLight 655-B3 Dyomics-680SeTau 425 BODIPY FL DyLight 655-B4 Dyomics-681 SeTau 647 BODIPY FL-XDyLight 662Q Dyomics-682 Square 635 BODIPY R6G DyLight 675-B1Dyomics-700 Square 650 BODIPY TMR DyLight 675-B2 Dyomics-701 Square 660BODIPY TR DyLight 675-B3 Dyomics-703 Square 672 C5.5 DyLight 675-B4Dyomics-704 Square 680 C7 DyLight 679-C5 Dyomics-730 Sulforhodamine 101CAL Fluor Gold 540 DyLight 680 Dyomics-731 TAMRA CAL Fluor Green 510DyLight 683Q Dyomics-732 TET CAL Fluor Orange 560 DyLight 690-B1Dyomics-734 Texas Red CAL Fluor Red 590 DyLight 690-B2 Dyomics-749 TMRCAL Fluor Red 610 DyLight 696Q Dyomics-749P1 TRITC CAL Fluor Red 615DyLight 700-B1 Dyomics-750 Yakima Yellow CAL Fluor Red 635 DyLight730-B1 Dyomics-751 Zenon Cascade Blue DyLight 730-B2 Dyomics-752 Zy3CF350 DyLight 730-B3 Dyomics-754 Zy5 CF405M DyLight 730-B4 Dyomics-776Zy5.5 CF405S DyLight 747 Dyomics-777 Zy7 CF488A DyLight 747-B1Dyomics-778 CF514 DyLight 747-B2 Dyomics-780

In certain embodiments, the luminescent marker may be (Dye 101) or (Dye102), of formulae:

or an analog thereof. In some embodiments, each sulfonate or carboxylateis independently optionally protonated. In some embodiments, the dyesabove are attached to the linker or nucleotide by formation of an amidebond at the indicated point of attachment.

In certain embodiments, at least one type, at least two types, at leastthree types, or at least four of the types of luminescently labelednucleotides comprise a luminescent marker selected from the groupconsisting of 6-TAMRA, 5/6-Carboxyrhodamine 6G, Alex Fluor 546, AlexaFluor 555, Alexa Fluor 568, Alexa Fluor 610, Alexa Fluor 647, AberriorStar 635, ATTO 647N, ATTO Rho14, Chromis 630, Chromis 654A, Chromeo 642,CF514, CF532, CF543, CF546, CF546, CF555, CF568, CF633, CF640R, CF660C,CF660R, CF680R, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Dyomics-530,Dyomics-547P1, Dyomics-549P1, Dyomics-550, Dyomics-554, Dyomics-555,Dyomics-556, Dyomics-560, Dyomics-650, Dyomics-680, DyLight 554-R1,DyLight 530-R2, DyLight 594, DyLight 635-B2, DyLight 650, DyLight655-B4, DyLight 675-B2, DyLight 675-B4, DyLight 680, HiLyte Fluor 532,HiLyte Fluor 555, HiLyte Fluor 594, LightCycler 640R, Seta 555, Seta670, Seta700, SeTau 647, and SeTau 665, or are of formulae (Dye 101) or(Dye 102), as described herein.

In some embodiments, at least one type, at least two types, at leastthree types, or at least four of the types of luminescently labelednucleotides comprise a luminescent marker selected from the groupconsisting of Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, AlexaFluor 594, Alexa Fluor 610, CF532, CF543, CF555, CF594, Cy3, DyLight530-R2, DyLight 554-R1, DyLight 590-R2, DyLight 594, and DyLight 610-B1,or are of formulae (Dye 101) or (Dye 102).

In some embodiments, a first and second type of luminescently labelednucleotide comprise a luminescent marker selected from the groupconsisting of Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, CF532,CF543, CF555, Cy3, DyLight 530-R2, and DyLight 554-R1, and a third andfourth type of luminescently labeled nucleotide comprise a luminescentmarker selected from the group consisting of Alexa Fluor 594, AlexaFluor 610, CF594, DyLight 590-R2, DyLight 594, and DyLight 610-B1, orare of formulae (Dye 101) or (Dye 102).

E. Linkers

A luminescent marker may be attached to the molecule directly, e.g., bya bond, or may be attached via a linker. In certain embodiments, thelinker comprises one or more phosphates. In some embodiments, anucleoside is connected to a luminescent marker by a linker comprisingone or more phosphates. In some embodiments, a nucleoside is connectedto a luminescent marker by a linker comprising three or more phosphates.In some embodiments, a nucleoside is connected to a luminescent markerby a linker comprising four or more phosphates.

In certain embodiments, a linker comprises an aliphatic chain. In someembodiments a linker comprises —(CH₂)_(n)—, wherein n is an integer from1 to 20, inclusive. In some embodiments, n is an integer from 1 to 10,inclusive. In certain embodiments, a linker comprises a heteroaliphaticchain. In some embodiments, a linker comprises a polyethylene glycolmoiety. In some embodiments, a linker comprises a polypropylene glycolmoiety. In some embodiments, a linker comprises —(CH₂CH₂O)_(n)—, whereinn is an integer from 1 to 20, inclusive. In some embodiments, a linkercomprises —(CH₂CH₂O)_(n)—, wherein n is an integer from 1 to 10,inclusive. In certain embodiments, a linker comprises —(CH₂CH₂O)₄—. Incertain embodiments, a linker comprises one or more arylenes. In someembodiments, a linker comprises one or more phenylenes (e.g.,para-substituted phenylene). In certain embodiments, a linker comprisesa chiral center. In some embodiments, a linker comprises proline, or aderivative thereof. In some embodiments, a linker comprises a prolinehexamer, or a derivative thereof. In some embodiments, a linkercomprises coumarin, or a derivative thereof. In some embodiments, alinker comprises naphthalene, or a derivative thereof. In someembodiments, a linker comprises anthracene, or a derivative thereof. Insome embodiments, a linker comprises a polyphenylamide, or a derivativethereof. In some embodiments, a linker comprises chromanone, or aderivative thereof. In some embodiments, a linker comprises4-aminopropargyl-L-phenylalanine, or a derivative thereof. In certainembodiments, a linker comprises a polypeptide.

In some embodiments, a linker comprises an oligonucleotide. In someembodiments, a linker comprises two annealed oligonucleotides. In someembodiments, the oligonucleotide or oligonucleotides comprisedeoxyribose nucleotides, ribose nucleotide, or locked ribosenucleotides. In certain embodiments, a linker comprises aphotostabilizer.

F. Sample Well Surface Preparation

In certain embodiments, a method of detecting one or luminescentlylabeled molecule is performed with the molecules confined in a targetvolume. In some embodiments, the target volume is a region within asample well (e.g., a nanoaperture). In certain embodiments, the samplewell comprises a bottom surface comprising a first material andsidewalls formed by a plurality of metal or metal oxide layers. In someembodiments, the first material is a transparent material or glass. Insome embodiments, the bottom surface is flat. In some embodiments, thebottom surface is a curved well. In some embodiments, the bottom surfaceincludes a portion of the sidewalls below the sidewalls formed by aplurality of metal or metal oxide layers. In some embodiments, the firstmaterial is fused silica or silicon dioxide. In some embodiments, theplurality of layers each comprise a metal (e.g., Al, Ti) or metal oxide(e.g., Al₂O₃, TiO₂, TiN).

G. Passivation

In some embodiments when one or more molecule or complex is immobilizedon a surface, it is desirable to passivate other surfaces of the deviceto prevent immobilization at an undesired location. In some embodiments,the molecule or complex is immobilized on a bottom surface of a samplewell and the sidewalls of the sample well are passivated. In someembodiments, the sidewalls are passivated by the steps of: depositing ametal or metal oxide barrier layer on the sidewall surfaces; andapplying a coating to the barrier layer. In some embodiments, the metaloxide barrier layer comprises aluminum oxide. In some embodiments, thestep of depositing comprises depositing the metal or metal oxide barrierlayer on the sidewall surfaces and the bottom surface. In someembodiments, the step of depositing further comprises etching metal ormetal oxide barrier layer off of the bottom surface.

In some embodiments, the barrier layer coating comprises phosphonategroups. In some embodiments, the barrier layer coating comprisesphosphonate groups with an alkyl chain. In some embodiments, the barrierlayer coating comprises a polymeric phosphonate. In some embodiments,the barrier layer coating comprises polyvinylphosphonic acid (PVPA). Insome embodiments, the barrier layer coating comprises phosphonate groupswith a substituted alkyl chain. In some embodiments, the alkyl chaincomprises one or more amides. In some embodiments, the alkyl chaincomprises one or more poly(ethylene glycol) chains. In some embodiments,the coating comprises phosphonate groups of formula:

wherein n is an integer between 0 and 100, inclusive, and

is hydrogen or a point of attachment to the surface. In some embodimentsn is an integer between 3 and 20, inclusive. In some embodiments, thebarrier layer coating comprises a mixture of different types ofphosphonate groups. In some embodiments, the barrier layer coatingcomprises a mixture of phosphonate groups comprising poly(ethyleneglycol) chains of different PEG weight.

In certain embodiments, the barrier layer comprises nitrodopa groups. Incertain embodiments, the barrier layer coating comprises groups offormula:

wherein R^(N) is an optionally substituted alkyl chain and

is hydrogen or a point of attachment to the surface. In someembodiments, R^(N) comprises a polymer. In some embodiments, R^(N)comprises a poly(lysine) or a poly(ethylene glycol). In someembodiments, the barrier layer comprises a co-polymer of poly(lysine)comprising lysine monomers, wherein the lysine monomers independentlycomprise PEG, nitrodopa groups, phosphonate groups, or primary amines.In certain embodiments, the barrier layer comprises a polymer of formula(P):

In some embodiments, X is —OMe, a biotin group, phosphonate, or silane.In some embodiments, each of i, j, k, and l is independently an integerbetween 0 and 100, inclusive.

H. Polymerase Immobilization

In some embodiments, when one or more molecule or complex is immobilizedon a surface the surface is functionalized to allow for attachment ofone or more of the molecules or complexes. In some embodiments, thefunctionalized surface is a bottom surface of a sample well. In certainembodiments, the functionalized surface comprises a transparent glass.In certain embodiments, the functionalized surface comprises fusedsilica or silicon dioxide. In some embodiments, the functionalizedsurface is functionalized with a silane. In some embodiments, thefunctionalized surface is functionalized with an ionically chargedpolymer. In some embodiments, the ionically charged polymer comprisespoly(lysine). In some embodiments, the functionalized surface isfunctionalized with poly(lysine)-graft-poly(ethylene glycol). In someembodiments, the functionalized surface is functionalized withbiotinlyated bovine serum albumin (BSA).

In certain embodiments, the functionalized surface is functionalizedwith a coating comprising nitrodopa groups. In certain embodiments, thecoating comprises groups of formula:

wherein R^(N) is an optionally substituted alkyl chain and

is hydrogen or a point of attachment to the surface. In someembodiments, R^(N) comprises a polymer. In some embodiments, R^(N)comprises a poly(lysine) or a poly(ethylene glycol). In someembodiments, R^(N) comprises a biotinylated poly(ethylene glycol). Insome embodiments, the coating comprises a co-polymer of poly(lysine)comprising lysine monomers, wherein the lysine monomers independentlycomprise PEG, biotinylated PEG, nitrodopa groups, phosphonate groups, orsilanes. In certain embodiments, the coating comprises a polymer offormula (P):

In some embodiments, X is —OMe, a biotin group, phosphonate, or silane.In some embodiments, each of i, j, k, and l is independently an integerbetween 0 and 100, inclusive.

In some embodiments, the functionalized surface is functionalized with asilane comprising an alkyl chain. In some embodiments, thefunctionalized surface is functionalized with a silane comprising anoptionally substituted alkyl chain. In some embodiments, the surface isfunctionalized with a silane comprising a poly(ethylene glycol) chain.In some embodiments, the functionalized surface is functionalized with asilane comprising a coupling group. For example the coupling group maycomprise chemical moieties, such as amine groups, carboxyl groups,hydroxyl groups, sulfhydryl groups, metals, chelators, and the like.Alternatively, they may include specific binding elements, such asbiotin, avidin, streptavidin, neutravidin, lectins, SNAP-tags™ orsubstrates therefore, associative or binding peptides or proteins,antibodies or antibody fragments, nucleic acids or nucleic acid analogs,or the like. Additionally, or alternatively, the coupling group may beused to couple an additional group that is used to couple or bind withthe molecule of interest, which may, in some cases include both chemicalfunctional groups and specific binding elements. By way of example, acoupling group, e.g., biotin, may be deposited upon a substrate surfaceand selectively activated in a given area. An intermediate bindingagent, e.g., streptavidin, may then be coupled to the first couplinggroup. The molecule of interest, which in this particular example wouldbe biotinylated, is then coupled to the streptavidin.

In some embodiments, the functionalized surface is functionalized with asilane comprising biotin, or an analog thereof. In some embodiments, thesurface is functionalized with a silane comprising a poly(ethylene)glycol chain, wherein the poly(ethylene glycol) chain comprises biotin.In certain embodiments, the functionalized surface is functionalizedwith a mixture of silanes, wherein at least one type of silane comprisesbiotin and at least one type of silane does not comprise biotin. In someembodiments, the mixture comprises about 10 fold less, about 25 foldless, about 50 fold less, about 100 fold less, about 250 fold less,about 500 fold less, or about 1000 fold less of the biotinylated silanethan the silane not comprising biotin.

FIG. 10-3 depicts a non-limiting exemplary process for preparing thesample well surface from the fabricated chip (e.g., integrated device)to initiation of a sequencing reaction. The sample well is depicted witha bottom surface (unshaded rectangle) and sidewalls (shaded verticalrectangles). The sidewalls may be comprised of multiple layers (e.g.,Al, Al₂O₃, Ti, TiO₂, TiN). In step (a) the sidewalls are deposited witha barrier layer of Al₂O₃. The Al₂O₃ barrier layer is then coated, instep (b), with a PEG phosphonate groups, for example, by treating thesurface with one or more PEG-phosphonic acids. In step (c), the bottomsurface is functionalized, for example, with a mixture of PEG-silane andbiotinylated-PEG-silane. The ovals represent individual biotin groupswhich may provide sites for an attachment of a single molecule orcomplex, such as a polymerase complex. In step (d), a polymerase complexis attached to a biotin group on the bottom surface. The polymerase maybe attached by way of a binding agent, such as streptavidin, and abiotin tag on the polymerase complex. The polymerase complex may furthercomprise a template nucleic acid and primer (not shown). Step (e)depicts the initiation of a sequencing reaction by exposure of theimmobilized polymerase complex to luminescently labeled nucleotides.

I. Polymerases

The term “polymerase,” as used herein, generally refers to any enzyme(or polymerizing enzyme) capable of catalyzing a polymerizationreaction. Examples of polymerases include, without limitation, a nucleicacid polymerase, a transcriptase or a ligase. A polymerase can be apolymerization enzyme.

Embodiments directed towards single molecule nucleic acid extension(e.g., for nucleic acid sequencing) may use any polymerase that iscapable of synthesizing a nucleic acid complementary to a target nucleicacid molecule. In some embodiments, a polymerase may be a DNApolymerase, an RNA polymerase, a reverse transcriptase, and/or a mutantor altered form of one or more thereof.

Embodiments directed towards single molecule nucleic acid sequencing mayuse any polymerase that is capable of synthesizing a nucleic acidcomplementary to a target nucleic acid. Examples of polymerases include,but are not limited to, a DNA polymerase, an RNA polymerase, athermostable polymerase, a wild-type polymerase, a modified polymerase,E. coli DNA polymerase I, T7 DNA polymerase, bacteriophage T4 DNApolymerase φ29 (psi29) DNA polymerase, Taq polymerase, Tth polymerase,Tli polymerase, Pfu polymerase, Pwo polymerase, VENT polymerase,DEEPVENT polymerase, EX-Taq polymerase, LA-Taq polymerase, Ssopolymerase, Poc polymerase, Pab polymerase, Mth polymerase, ES4polymerase, Tru polymerase, Tac polymerase, Tne polymerase, Tmapolymerase, Tca polymerase, Tih polymerase, Tfi polymerase, Platinum Taqpolymerases, Tbr polymerase, Tfl polymerase, Tth polymerase, Pfutubopolymerase, Pyrobest polymerase, Pwo polymerase, KOD polymerase, Bstpolymerase, Sac polymerase, Klenow fragment, polymerase with 3′ to 5′exonuclease activity, and variants, modified products and derivativesthereof. In some embodiments, the polymerase is a single subunitpolymerase. Non-limiting examples of DNA polymerases and theirproperties are described in detail in, among other places, DNAReplication 2nd edition, Kornberg and Baker, W. H. Freeman, New York,N.Y. (1991).

Upon base pairing between a nucleobase of a target nucleic acid and thecomplementary dNTP, the polymerase incorporates the dNTP into the newlysynthesized nucleic acid strand by forming a phosphodiester bond betweenthe 3′ hydroxyl end of the newly synthesized strand and the alphaphosphate of the dNTP. In examples in which the luminescent markerconjugated to the dNTP is a fluorophore, its presence is signaled byexcitation and a pulse of emission is detected during or after the stepof incorporation. For detection markers that are conjugated to theterminal (gamma) phosphate of the dNTP, incorporation of the dNTP intothe newly synthesized strand results in release the beta and gammaphosphates and the detection marker, which is free to diffuse in thesample well, resulting in a decrease in emission detected from thefluorophore.

In some embodiments, the polymerase is a polymerase with highprocessivity. However, in some embodiments, the polymerase is apolymerase with reduced processivity. Polymerase processivity generallyrefers to the capability of a polymerase to consecutively incorporatedNTPs into a nucleic acid template without releasing the nucleic acidtemplate.

In some embodiments, the polymerase is a polymerase with low 5′-3′exonuclease activity and/or 3′-5′ exonuclease. In some embodiments, thepolymerase is modified (e.g., by amino acid substitution) to havereduced 5′-3′ exonuclease activity and/or 3′-5′ activity relative to acorresponding wild-type polymerase. Further non-limiting examples of DNApolymerases include 9° Nm™ DNA polymerase (New England Biolabs), and aP680G mutant of the Klenow exo− polymerase (Tuske et al. (2000) JBC275(31):23759-23768). In some embodiments, a polymerase having reducedprocessivity provides increased accuracy for sequencing templatescontaining one or more stretches of nucleotide repeats (e.g., two ormore sequential bases of the same type).

Embodiments directed toward single molecule RNA extension (e.g., for RNAsequencing) may use any reverse transcriptase that is capable ofsynthesizing complementary DNA (cDNA) from an RNA template. In suchembodiments, a reverse transcriptase can function in a manner similar topolymerase in that cDNA can be synthesized from an RNA template via theincorporation of dNTPs to a reverse transcription primer annealed to anRNA template. The cDNA can then participate in a sequencing reaction andits sequence determined as described above and elsewhere herein. Thedetermined sequence of the cDNA can then be used, via sequencecomplementarity, to determine the sequence of the original RNA template.Examples of reverse transcriptases include Moloney Murine Leukemia Virusreverse transcriptase (M-MLV), avian myeloblastosis virus (AMV) reversetranscriptase, human immunodeficiency virus reverse transcriptase(HIV-1) and telomerase reverse transcriptase.

The processivity, exonuclease activity, relative affinity for differenttypes of nucleic acid, or other property of a nucleic acid polymerasecan be increased or decreased by one of skill in the art by mutation orother modification relative to a corresponding wild-type polymerase.

J. Lifetime Measurements

Lifetime measurements may be performed using one excitation energywavelength to excite a marker in a sample well. A combination of markershaving distinct lifetimes are selected to distinguish among theindividual markers based on the lifetime measurements. Additionally, thecombination of markers are able to reach an excited state whenilluminated by the excitation source used. An integrated deviceconfigured for lifetime measurements using one excitation may includemultiple pixels positioned along a row where each sample well isconfigured to couple with the same waveguide. A pixel includes a samplewell and a sensor. One or more microcavities or a bullseye grating maybe used to couple the waveguide to the sample well for each pixel.

A pulsed excitation source may be one of the pulsed excitation sourcesusing the techniques described above. In some instances, the pulsedexcitation source may be a semiconductor laser diode configured to emitpulses through direct modulated electrical pumping of the laser diode.The power of the pulses is less than 20 dB of the pulse peak power atapproximately 250 picoseconds after the peak. The time interval for eachexcitation pulse is in the range of 20-200 picoseconds. The timeduration between each excitation pulse is in the range of 1-50nanoseconds. A schematic of how example measurements may be performed isshown in FIG. 10-4. Since one excitation energy is used, an excitationfilter suitable for reducing transmission of the excitation energy tothe sensor may be formed in the integrated device, such as thewavelength excitation filter discussed above.

The sensor for each pixel has at least one photosensitive region per apixel. The photosensitive region may have dimensions of 5 microns by 5microns. Photons are detected within time intervals of when they reachthe sensor. Increasing the number of time bins may improve resolution ofthe recorded histogram of photons collected over a series of time binsand improve differentiation among different luminescent markers. In someembodiments, focusing elements may be integrated with the sensor inorder to improve collection of photons emitted by a marker in anassociated sample well. Such focusing elements may include a Fresnellens as shown in FIG. 10-5. When the sensor is configured to detect aparticular wavelength, the four luminescent markers may emitluminescence similar to the particular wavelength. Alternatively, thefour luminescent markers may emit luminescence at different wavelengths.

An example set of four luminescent markers that are distinguishablebased on lifetime measurements are ATRho14, Cy5, AT647N, and CF633 asshown by the plot in FIG. 10-6. These four markers have varyinglifetimes and produce distinguishing histograms when at least four timebins are used. FIG. 10-7 outlines a signal profile for each of thesemarkers across 16 time bins. The signal profile is normalized for eachmarker. The time bins vary in time interval in order to provide a uniquesignal profile for each of the markers. FIGS. 10-8 and 10-9 illustratessignal profiles, both continuous and discrete, respectively, of anotherexemplary set of markers, ATTO Rho14, D650, ST647, and CF633, that aredistinguishable based on lifetime measurements. Other sets of markersinclude ATTO Rho14, C647, ST647, CF633; Alexa Fluor647, B630, C640R,CF633; and ATTO Rho14, ATTO 647N, AlexaFluor647, CF633.

K. Spectral-Lifetime Measurements

Lifetime measurements may be combined with spectral measurements of oneor more luminescent markers. Spectral measurements may depend on thewavelength of luminescence for individual markers and are captured usingat least two sensor regions per pixel. An exemplary structure of theintegrated device includes pixels that each have a sensor with twodistinct regions, each region configured to detect a differentwavelength. A multi-wavelength filter, such as the one shown anddescribed in FIG. 7-7, may be used to selectively transmit light ofdifferent wavelength to each sensor region. For example, one sensorregion and filter combination may be configured to detect red lightwhile another sensor region and filter combination may be configured todetect green light.

Combining both lifetime measurements with spectral measurements may beperformed using one excitation energy wavelength to excite a marker in asample well. A combination of markers is selected having at least twodistinct luminescence wavelengths where the markers emitting at awavelength have distinct lifetimes are selected to distinguish among theindividual markers based on the lifetime and spectral measurements.Additionally, the combination of markers is selected to be able to reachan excited state when illuminated by the excitation source used.

The excitation source is a pulsed excitation source, and may be one ofthe excitation sources using the techniques described above. In someinstances, the pulsed excitation source may be a semiconductor laserdiode configured to emit pulses through direct modulated electricalpumping of the laser diode. The power of the pulses are less than 20 dBthe peak power after 250 picoseconds after the peak. The time durationfor each excitation pulse is in the range of 20-200 picoseconds. Thetime interval between each excitation pulse is in the range of 1-50nanoseconds. A schematic of how example measurements may be performed isshown in FIG. 10-10. Since one excitation energy is used, an excitationfilter suitable for reducing transmission of the excitation energy tothe sensor may be used, such as the wavelength excitation filterdiscussed above with reference to FIG. 7-5.

The sensor for each pixel has at least two photosensitive regions perpixel. In some embodiments there are two photosensitive regions per apixel. In other embodiments, there are four photosensitive regions per apixel. Each photosensitive region is configured to detect a differentwavelength or range of wavelengths. Photons are detected within timeintervals of when they reach the sensor. Increasing the number of timebins may improve resolution of the recorded histogram of photonscollected over a series of time bins and improve differentiation amongdifferent luminescent markers by their individual lifetimes. In someembodiments, there are two time bins per a region of the sensor. Inother embodiments, there are four time bins per a region of the sensor.

An example set of four luminescent markers that are distinguishablebased on lifetime measurements are ATTO Rho14, AS635, Alexa Fluor647,and ATTO 647N. These four markers have two that emit at one similarwavelength and another similar wavelength. Within each pair of markersthat emit at a similar wavelength, the pair of markers have differentlifetimes and produce distinguishing histograms when at least four timebins are used. In this example, ATTO Rho14 and AS635 emit similarluminescence wavelengths and have distinct lifetimes. Alexa Fluor 647and ATTO 647N emit similar luminescence wavelengths, different from thewavelengths emitted by ATTO Rho 14 and AS635, and have distinctlifetimes. FIG. 10-11 shows a plot lifetime as a function of emissionwavelength for this set of markers to illustrate how each of thesemarkers is distinguishable based on a combination of lifetime andemission wavelength. FIG. 10-12 shows a plot of power as a function ofwavelength for ATT Rho14, Alexa Fluor 647, and ATT) 647N. FIG. 10-13shows plots of fluorescence signal over time for each one of thesemarkers when present in a sample well with a diameter of 135 nm. FIG.10-14 illustrates the signal profile for these markers across fourphotosensitive regions and each region captures four time bins. Thesignal profiles are normalized and are used to distinguish among thedifferent markers by the relative number of photons captured by aphotosensitive region for each of the four time bins. Other sets of fourfluorophores for such spectral-lifetime measurements are ATRho14, D650,ST647, CF633; ATTO Rho14, C647, ST647, CF633; Alexa Fluor 647, B630,C640R, CF633; and ATTO Rho 14, ATTO 647N, Alexa Fluor 647, CF633. FIG.10-15 shows a plot of the signal profile of intensity over time forATRho14, D650, ST647, and C633. FIG. 10-16 illustrates the signalprofile for ATRho14.

L. Lifetime-Excitation Energy Measurements

Lifetime measurements combined with using at least two excitation energywavelengths may be used to distinguish among multiple markers. Somemarkers may excite when one excitation wavelength is used and notanother. A combination of markers having distinct lifetimes is selectedfor each excitation wavelength to distinguish among the individualmarkers based on the lifetime measurements. In this embodiment, anintegrated device may be configured to have each pixel with a sensorhaving one region and the external excitation source may be configuredto provide two excitation energy wavelengths are electrically modulatedpulsed diode lasers with temporal interleaving.

The excitation source is a combination of at least two excitationenergies. The excitation source is a pulsed excitation source and may beone or more of the excitation sources using the techniques describedabove. In some instances, the pulsed excitation source may be twosemiconductor laser diode configured to emit pulses through directmodulated electrical pumping of the laser diode. The power of the pulsesare 20 dB less than the pulse peak power at 250 picoseconds after thepeak. The time interval for each excitation pulse is in the range of20-200 picoseconds. The time interval between each excitation pulse isin the range of 1-50 nanoseconds. One excitation wavelength is emittedper a pulse and by knowing the excitation wavelength a subset of markerswith distinct lifetimes is uniquely identified. In some embodiments,pulses of excitation alternate among the different wavelengths. Forexample, when two excitation wavelengths are used subsequent pulsesalternate between one wavelength and the other wavelength. A schematicof how example measurements may be performed is shown in FIG. 10-17. Anysuitable technique for combining multiple excitation sources andinterleaving pulses having different wavelengths may be used. Examplesof some techniques for delivering pulses of more than one excitationwavelength to a row of sample wells are illustrated are describedherein. In some embodiments, there is a single waveguide per a row ofsample wells and there are two excitation sources that are combined suchthat pulses of excitation energy alternate between the two excitationwavelengths. In some embodiments, there are two waveguides per a row ofsample wells and each waveguide is configured to carry one of twoexcitation wavelengths. In other embodiments, there is a singlewaveguide per a row of pixels and one wavelength couples to one end ofthe waveguide and another wavelength couples to the other end.

The sensor for each pixel has at least one photosensitive region per apixel. The photosensitive region may have dimensions of 5 microns by 5microns. Photons are detected within time intervals of when they reachthe sensor. Increasing the number of time bins may improve resolution ofthe recorded histogram of photons collected over a series of time binsand improve differentiation among different luminescent markers. Thesensor has at least two time bins.

An example set of four luminescent markers that are distinguishablebased on lifetime measurements are Alexa Fluor 546, Cy3B, Alexa Fluor647, and ATTO 647N. As shown in FIG. 10-18, Alexa Fluor 546 and Cy3Bexcite at one wavelength, such as 532 nm, and have distinct lifetimes.Alexa Fluor 647 and ATTO 647N excite at another wavelength, 640 nm, andhave distinct lifetimes as shown in FIG. 10-19. Distinguishablenormalized signal profiles across 16 time bins for ATTO647N and CF633,which are both excited at 640 nm, are shown in FIG. 10-20. By detectinga photon after a known excitation wavelength, one of these two pairs ofmarkers may be determined based on the previous excitation wavelengthand each marker for a pair is identified based on lifetime measurements.

VI. Fabrication Steps

The above integrated device may be fabricated in any suitable way. Whatfollows is a description of the fabrication of various components of theintegrated device, which may be combined in any way with techniquesknown in the art, to create a suitable integrated device.

A. Sample Well Fabrication Process

A sample well (e.g., nanoaperature) may be fabricated in any suitableway. In some embodiments, a sample well may be fabricated using standardphotolithography processes and etching techniques. A sample well may beformed in a layer having a metal (e.g., Al, TiN) or any suitablematerial compatible with photolithography processing. FIG. 11-1illustrates an exemplary method for fabricating a sample well of anintegrated device. Layer 11-112 forms a sample well and may include ametal such as Al or TiN. Layer 11-110 may act as a dielectric layer andmay be formed from any suitable dielectric substrate, such as SiO₂ orsilicon nitride. One step of the method includes depositing layer 11-112directly on substrate 11-110. In some embodiments, additional layers maybe deposited between layer 11-112 and layer 11-110. Layer 11-112 may bedeposited at any suitable thickness, and in some embodiments, thethickness may determine the height of the resulting sample well. Thethickness of layer 11-112 may be approximately 50 nm, approximately 100nm, or approximately 150 nm. An anti-reflection coating (ARC) 11-122 isthen deposited on top of layer 11-112. Etch mask 11-120 (e.g.,photoresist etch mask) is deposited on ARC 11-122. Conventionalphotolithographic techniques are used to pattern a hole in etch mask11-120 and ARC layer 11-122. The hole patterned by etch mask 11-120 mayhave a diameter of approximately 50 nm, approximately 100 nm, orapproximately 150 nm. The hole pattern is then transferred to underlyinglayer 11-112 using an etch, for example, reactive ion etchingtechniques, to form the sample well. Etching may stop at the surface oflayer 11-110, or etching may create a divot in layer 11-110 under thehole in layer 11-112. Conventional techniques are used to strip etchmask 11-120 and ARC 11-122 off of layer 11-112. The sample well may havea diameter oft approximately 50 nm, approximately 100 nm, orapproximately 150 nm.

Alternatively, a sample well may be fabricated using standardphotolithography processes and lift-off techniques. FIG. 11-2illustrates an exemplary method of forming a sample well using lift-offtechniques. Sample well is formed in layer 11-212, which may include ametal (e.g., Al, Au, Cr). Layer 11-212 is formed over substrate layer11-210, which may include any suitable material such as a dielectric(e.g., SiO₂). Deposition of layer 11-212 may occur separately from andafter the photolithographic process. The first step in the lift-offfabrication process shown in FIG. 11-2 may involve depositinganti-reflection coating (ARC) 11-222 on substrate 11-210 followed byphotoresist etch mask 11-220 directly on top of substrate 11-210.Conventional photolithographic techniques are used to pattern thephotoresist such that a pillar 11-230 of resist is left behind. Thepillar may have any suitable size and shape that may correspond to aresulting sample well. The pillar may have a diameter of approximately50 nm, approximately 100 nm, or approximately 150 nm. Such techniquesmay include dissolving the resist and the ARC layer around the pillaroff of the substrate. The following step may involve depositing layer11-212 directly on top of the pillar of resist and the substrate,creating a capped pillar. In other embodiments, additional layers may bedeposited before or after deposition of layer 11-212. As a non-limitingexample, TiN may be deposited on layer 11-212 formed of Al, optionallyfollowed by a deposition of Al₂O₃. Layer 11-212 may be deposited at anysuitable thickness, and in some embodiments may have a thickness ofapproximately 50 nm, approximately 100 nm, or approximately 150 nm. Toform the sample well, the capped pillar may be stripped by a solvent inthe case that photoresist is used or by selective etching in the casethat silicon dioxide or silicon nitride hard etch masks are used. Thesample well may have a diameter oft approximately 50 nm, approximately100 nm, or approximately 150 nm.

Alternatively, a sample well may be fabricated using standardphotolithography processes and an alternate lift-off technique. FIG.11-3 illustrates an exemplary embodiment of forming a sample well of anintegrated device. A hard etch mask layer 11-314 is deposited onsubstrate 11-310. Hard etch mask layer 11-314 may include Ti or anyother suitable material. Substrate 11-310 may include a dielectric(e.g., SiO₂) or any other suitable material. A layer of ARC 11-322 isthen deposited onto the hard etch mask layer 11-314 followed byphotoresist layer 11-320. Conventional photolithographic techniques areused to pattern the photoresist such that a pillar 11-320 of resist isformed. This photoresist pillar pattern is used as an etch mask foretching the ARC layer 11-322 and hard etch mask layer 11-314.Photoresist layer 11-320 and ARC 11-322 is then stripped, and pillar11-330 of hard etch mask is left behind. Conventional techniques may beused to dissolve the remaining photoresist and the ARC layer off thepillar. The following step may involve depositing layer 11-312 directlyon top of pillar 11-330 creating a capped pillar. To form the samplewell, the capped pillar is stripped by a hydrogen peroxide etch, orother suitable etch, which erodes layer 11-314, “lifts off” the cap andresults in a sample well in layer 11-312.

In some embodiments, the sample well may be fabricated to attenuateplasmon transmission through the sample well in any suitable way. Forexample, the sample well may be fabricated in a multi-layered stack. Themulti-layered stack may include, but is not limited to, a metal layerdeposited on a substrate, an absorbing layer and/or a surface layer. Thesurface layer may be a passivation layer. The multi-layer stack may befabricated in any suitable way. Conventional patterning and etchingtechniques may be used. A metal layer may be deposited onto a substrate.Alternatively, an absorbing layer may be deposited onto the metal layer.Alternatively, a surface passivation layer may be deposited onto theabsorbing layer/metal layer stack. A photoresist and anti-reflectionlayer may be deposited onto the top layer of the multilayer stack. Thephotoresist layer may be patterned with the dimensions of the samplewell. The multi-layer stack may be directly etched to form the samplewell.

The absorbing layers may include any suitable absorbing materials.Non-limiting examples include silicon nitride, TiN, aSi, TaN, Ge and/orCr. Variants of the named materials are also possible, such as Si₃N₄.The metal layer and the surface layer may be made of any suitablematerials. For example, Al, AlSi, or AlCu may be used for the metallayer. The surface layer may be made of Al or Al₂O₃, for example. Thesample well in the multilayer stack may be fabricating using theprocesses described above.

Additionally and/or alternatively, a reflective layer may be depositeddirectly on top of the substrate before depositing the multi-layeredstack to control the focus of the light beam during photolithography. Areflective layer may be deposited directly on top of the substrate andpatterned with the dimensions of the sample well. Optionally, a layer ofanti-reflection coating followed by a layer of photoresist may bedeposited on top of the patterned reflective coating layer and patternedto leave a pillar of ARC and photoresist at a location on the substrate.A multilayer stack may then be deposited on top of the pillar,reflective layer, and substrate. The capped pillar may be removed usinga lift-off process, as described above, forming a sample well at thelocation of the substrate where the piller had been.

Similarly, a Ti pillar may be used to create the sample well. The firststep may involve depositing a layer of Ti on the substrate, followed bya layer of anti-reflection coating and a layer of photoresist. The Tilayer may be patterned and etched to form a Ti pillar. The multilayerstack may be deposited on top of the Ti pillar and substrate. Finally,the Ti pillar may be removed forming a sample well at a location of thesubstrate corresponding to where the Ti pillar had been.

Any suitable deposition methods may be used. For example, PVD, CVD,sputtering, ALD, e-beam deposition and/or thermal evaporation may beused to deposit one or more layers. The deposition environment may becontrolled to prevent oxidation of the layers in between depositions.For example, the environment may be kept at a high vacuum and/orlow-oxygen state during and between depositions of one or more layers.

B. Sample Well Layer with Dips

The sample well layer may include dips such than the sample well (e.g.,nanoaperture) is positioned at a certain distance to the waveguide. Anysuitable technique for forming dips in the sample well layer may beused. One technique includes grayscale lithography used to form a resistwith topography followed by etching to transfer the topography to theoxide layer. After the oxide layer has the dip topography, the samplewell and/or other structures may be formed. FIG. 11-5 illustrates anexemplary embodiment of forming a sample well layer with dips. Waveguide11-820 is formed within surrounding material layer 11-810, and resist11-830 is patterned on a surface of surrounding material layer 11-810.The patterning of resist 11-830 may have any suitable size or shape. Thesurface of layer 11-810 is etched to form a desired dip shape based onthe patterning of resist 11-830. Sample well layer 11-812 is depositedon the etched surface of layer 11-810 by any combination of thetechniques described herein to form a sample well layer 11-810 with aportion having sample well 11-832 positioned a distance from waveguide11-820 to provide suitable coupling between waveguide 11-820 and samplewell 11-832.

Another technique for forming a sample well layer with dips may includeusing grayscale lithography to form topography into an oxide layer toexpose the waveguide and then oxide is deposited with a controlledthickness, followed by formation of the sample well and/or otherstructures. FIG. 11-6 illustrates an exemplary embodiment of forming asample well layer with dips. Waveguide 11-920 is formed withinsurrounding material layer 11-910. The surface of layer 11-910 is etchedto expose waveguide 11-920 using grayscale lithography. Layer 11-910 isreformed with controlled thickness. Components such as surface plasmonicstructures (not shown) may be formed. Sample well layer 11-912 formingsample well 11-932 is formed over layer 11-910.

In some embodiments, the entire array of sample wells are formed in adip region of the substrate. For example, the distance between thebottom metal layer of the sample well and the the waveguide may beapproximately 600 nm outside the region where the sample wells areformed, but may be reduced to approximately 350 nm where the sample wellarray is located. In this way, where a plurality of integrated devicesare being formed on a wafer, the location of each integrated device onthe wafer may be visually identified by the dip in the top surface ofthe wafer associated with each individual integrated device beingformed.

C. Concentric Grating (Bullseye) Fabrication Process

A concentric grating, or bullseye, may be fabricated in any suitableway. In some embodiments, a concentric grating may be fabricated usingstandard photolithography processes and etching techniques. Any suitabledielectric material, such as SiO₂ or silicon nitride, may be used toform the concentric grating. In the embodiment illustrated in FIG. 11-7,a SiO₂ layer 11-1010 is used to make the concentric grating. The firststep in the fabrication process may involve depositing a hard etch mask11-1014 directly on top of the SiO₂ layer 11-1010. The next step of11-1001 in the fabrication process may involve depositing a photoresistlayer 11-1020 directly on top of an anti-reflection coating (ARC) layer11-1022 onto the hard etch mask, which may include silicon. Conventionalphotolithographic techniques are used to create the bullseye pattern inthe hard etch mask such as by patterning the concentric grating into thephotoresist by step 11-1003 and etching the resist pattern into the ARClayer and hard etch mask by step 11-1005. The bullseye pattern is thentransferred to the underlying SiO₂ layer using etching, for examplereactive ion etching techniques to form the concentric grating by step11-1007. The thickness of the concentric grating can be any suitablethickness. In the embodiment illustrated in FIG. 11-7, the etch depth isapproximately 80 nm. Conventional techniques are used to strip theresist and etch mask residues and clean the surface of the concentricgrating by step 11-1009. The sample well in layer 11-1012 may befabricated directly on top of the concentric grating using the lift-offor etch processes by step 11-1011. In other embodiments, other layersmay be deposited between the concentric grating and the sample well.

Alternatively, in some embodiments, the sample well may be positionedcentral to the concentric grating. This precise alignment of the samplewell may be achieved in any suitable way. In the embodiment illustratedin FIG. 11-8, positioning of the sample well is achieved using aself-aligned fabrication process. The first step may involve forming theconcentric grating according to the techniques described above. However,in FIG. 11-8, a Ti hard etch mask 11-1114 is deposited on top of theSiO₂ substrate 11-1110 by step 11-1101. The bullseye pattern istransferred to the Ti layer using etching, for example reactive ionetching, by step 11-1103. A layer of resist 11-1120 and a layer ofanti-reflection coating (ARC) 11-1122 are deposited over the two centergaps in the Ti layer to cover the gaps and the center Ti pillar by step11-1105. The bullseye pattern is then transferred to the SiO₂ substrateusing conventional etching techniques to form the concentric grating bystep 11-1107. The Ti layer is then removed using an isotropic wet etchby step 11-1109, for example, using peroxide, but leaving the center Tipillar 11-1116 in place. The layer of resist is then stripped usingconventional techniques. The metal sample well layer is then depositedon top of the concentric grating and the Ti pillar. Lastly, themetal-capped Ti pillar is removed using a lift-off process leaving asample well precisely centered relative to the concentric grating bystep 11-1111.

The precise alignment of the sample well may be achieved in variousother ways. In the embodiment illustrated in FIG. 11-9, positioning ofthe sample well is achieved using an alternate self-aligned fabricationprocess. The first step may involve depositing the sample well layer(e.g., Al, Ti) 11-1212 directly on top of the SiO₂ concentric gratingsubstrate 11-1210 by step 11-1201. A hard etch mask 11-1214 may then bedeposited on top of layer 11-1212. The bullseye pattern is transferredto layer 11-1212 using conventional etching techniques by step 11-1203.A layer of resist 11-1220 and a layer of anti-reflection coating 11-1222are deposited over the center gap in layer 11-1212 to cover the positionwhere the sample well is to be formed by step 11-1205. The bullseyepattern is then transferred to the SiO₂ substrate using conventionaletching techniques to form the concentric grating by step 11-1207. Anadditional metal layer is deposited on top of the layer 11-1212 suchthat the metal fills the cavities in the SiO₂ substrate 11-1210 andcovers the resist layer 11-1214 by step 11-1209. In the embodimentillustrated in FIG. 11-9, Al is used as the additional metal layer butother suitable metals compatible with photolithographic processes may beused. Lastly, the metal-capped resist pillar 11-1230 is removed using alift-off process leaving a nanoaperture precisely centered relative tothe concentric grating by step 11-1211.

D. Microcavity Fabrication Process

A microcavity may be fabricated in any suitable way. In someembodiments, a microcavity may be fabricated using standardphotolithography processes and etching techniques. The microcavity mayinclude silicon nitride. The first step in the fabrication process mayinvolve depositing silicon nitride on an oxide film. The silicon nitridelayer may be patterned and etched to form the microcavity structure.After the silicon nitride is etched, an oxide is deposited over thesilicon nitride feature and polished flat, such as via CMP. The samplewell layer with a sample well may be fabricated above or near themicrocavity. The microcavity may be offset from the sample well. FIG.11-10 illustrates two possible fabrication designs for an offsetmicrocavity structure.

E. Reflector Layer Below Waveguide Grating Coupler

The reflector layer underneath the grating coupler may be formed in anysuitable way. The reflector layer may be a metal layer at a controlleddistance from the waveguide grating coupler in order to improveexcitation energy coupling into the waveguide. An exemplary fabricationprocess includes forming a recess in an oxide layer at the gratingcoupler location using lithography and/or etching. The reflectormaterial is deposited and fills the trench. A resist layer is formedover the reflector and lithography and etching is used to remove excessreflector material. Oxide is formed over the reflector, such as throughPECVD, to form a planar surface for waveguide fabrication.

F. Excitation Filter

An excitation filter may be formed by alternating layers of high and lowindex refractive material. Any suitable low refractive index materialsmay be used. Example low refractive index materials include silicondioxide formed using PVD, PECVD, LPCVD, ALD, and/or evaporationtechniques. Any suitable high refractive index material may be used.Example high refractive index materials include silicon, siliconnitride, titanium dioxide, and tantalum pentoxide.

G. Baffle

A baffle may be formed in a pixel between the sensor and the sample wellto block and/or absorb stray light, such as excitation light from thewaveguide, from being detected by the sensor. One technique is todeposit an absorbing layer over a raised section of the oxide layer,where the raised section overlays the sensor, followed by polishing viaCMP. Another technique is to deposit an absorptive thin film over theoxide layer and then use lithography and etching techniques to formholes in the absorptive layer over the sensor.

H. Lens Fabrication Process: Refractive Lens

A refractive lens array may be created in any suitable way to improveefficiency of focusing of excitation into and collection of emissionlight from the sample well. In some embodiments, a refractive lens arraymay be a “gapless” array to minimize “dead zones” on the lens array. Inthe embodiment illustrated in FIG. 11-11, a refractive microlens arrayis shown with no gaps between individual lenses. In some embodiments,fabricating a “gapless” array may involve two etching steps. A firstetch may establish the depth of the microlens topography by step11-1801. A second etch may follow the first etch to eliminate the planargaps between the individual microlenses by step 11-1803 such that onelens stops at the edge where another lens begins. The sum of the firstand second etches defines the focal length. In the embodimentillustrated in FIG. 11-12, a top view of a microlens array is shownafter the first HF etch (1), after the second HF etch (2), after themicrolens array is coated with a higher refractive index materialsilicon nitride (3), and after the high refractive index material ispolished and planarized (4).

Each refractive lens in the refractive lens array may be fabricated inany suitable way. An example refractive lens array is illustrated inFIG. 11-13 where a nanoaperture layer 11-2007 is fabricated on top of atransparent spacer layer 11-2001, which is on top of a dielectric lenslayer 11-2003, which is on top of a substrate 11-2005. In someembodiments, a refractive lens may be fabricated using standardphotolithography processes and etching techniques. Any suitabledielectric material, such as SiO₂ or silicon nitride, may be used toform the refractive lens. In the embodiment illustrated in FIG. 11-14,silicon nitride is used to fill in the SiO₂ substrate topography. Thefirst step 11-2101 in the fabrication process may involve depositing ahard etch mask 11-2114 directly on top of a SiO₂ substrate 11-2110. Anysuitable metal may be used for the hard etch mask 11-2114 that does notdissolve during the same etching process used for the SiO₂ layer. Forexample, Cr is used, but other metals are possible. The next step mayinvolve applying a photoresist layer 11-2120 on top of the hard etchmask 11-2114. Conventional photolithographic techniques are used tocreate a circular pattern in the hard etch mask by step 11-2103. Thecircular pattern is then transferred to the underlying Cr layer usingconventional etching techniques, such as reactive ion etchingtechniques, for example. The SiO₂ layer is etched using any suitableselective etching technique which can etch the SiO₂ but not the hardetch mask. For example, an isotropic wet etch using HF is used to createa concave surface in the SiO₂ layer. The hard etch mask 11-2114 is thenremoved using conventional etching techniques by step 11-2105.Optionally, a second wet etch using HF is performed to eliminate thegaps between lenses. To create the refractive lens, the cavity in theSiO₂ layer is filled with a high refractive index material layer11-2118, such as silicon nitride, by step 11-2107. Finally, the topsurface of the lens is planarized with conventional techniques by step11-2109, such as chemical mechanical polishing, for example. A spacerlayer 11-2124 may be deposited on top of the layer 11-2118 by step11-2111. For example, a spacer layer made of ORMOCER™ may be spun-coaton top of the silicon nitride layer. Alternatively, a layer of SiO₂ maybe deposited. The sample well may be fabricated directly on top of therefractive lens. In other embodiments, other layers may be depositedbetween the refractive lens and the sample well.

Alternatively, each refractive lens may include an anti-reflection layerto further improve optical efficiency. In some embodiments, ananti-reflection layer may coat a bottom, top, or all sides of a lens. Inthe embodiment illustrated in FIG. 11-15, an SiO₂ cavity 11-1810 is etchby step 11-1801, an anti-reflection layer 11-1822 is deposited on theetched cavity by step 11-1803, and a silicon nitride layer 11-1818 isdeposited to fill the cavity by st. The silicon nitride layer ispolished via CMP by step 11-1807 and a second antireflection layer11-1826 is deposited on top of the polished silicon nitride layer bystep 11-1809. Additional layers may be deposited on top of theantireflection layer, such as the spacer layer described above and shownas layer 11-1824 in FIG. 11-18. The anti-reflection layers may have thefollowing parameters: index of refraction, n_(C)=sqrt(n_(oxide),n_(nitride))=sqrt(1.46*1.91)=1.67; range of refractive index from 1.67to 1.75; and, thickness t=λ/(4*n_(C))=675 nm/(1.670*4)=101.1 nm. Theanti-reflection layer may be deposited in any suitable way. For example,PECVD may be used. Alternatively, LPCVD may be used.

I. Lens Fabrication Process: Fresnel Lens

A diffractive optical element (DOE) may have any suitable shape and maybe fabricated in any suitable way to improve the focusing ofluminescence on the CMOS sensors and the sorting of the luminescencephotons. In some embodiments, the DOE may include a section of a Fresnellens. As illustrated in FIGS. 11-16 to 11-20, the DOE is characterizedas a square section offset from the center of a Fresnel lens. Asillustrated in FIG. 11-17, the DOE may comprise two unit cell layerswhere the first layer 11-2301 contains “small” features and the secondlayer 11-2303 contains “large” features. The unit cell layers may haveany suitable pitch, and may further have varying pitch according to theoptical design of the Fresnel lens. As illustrated in the example inFIG. 11-17, the small DOE layer has a pitch of 220 nm and the large DOElayer has a pitch of 440 nm. The large DOE layer may be overlaid ontothe small DOE layer (or vice versa) to create a multilevel diffractiveoptic. FIG. 11-17 illustrates an example of an offset Fresnel array11-2405 where large fiducial markers surround the offset Fresnel lens.Additionally, the offset Fresnel array may be positioned on top of thesensor to provide focusing and spectral separation of luminescence intothe sensor.

Alternatively, a diffractive optical element (DOE) may be embeddedunderneath the nanoaperture to improve the focusing of the excitationenergy into and collection of luminescence from the nanoaperture. Insome embodiments, the embedded Fresnel lens positioned underneath thenanoaperture and the offset Fresnel lens positioned over the sensor mayhave a tiered structure with a variable period and variable step size.In other embodiments, only the offset Fresnel lens positioned over thesensor may have a variable period and variable step size. Thesediffractive lenses may be fabricated using standard photolithographyprocesses and etching techniques. As illustrated in FIG. 11-18, thediffractive lens pattern 11-2501 is characterized as having a tieredstructure comprising large steps (large pattern) and small steps (smallpattern) on each larger step, both having a decreasing period whenviewed from left to right. The fabrication process for thevariable-period stepped diffractive lens may involve etching the largesteps first, followed by etching the small steps as illustrated in FIG.11-19, which may protect the corners of the large steps during thesecond etch. An alternate approach is to etch the small steps first on aflat substrate, followed by etching the large steps, as illustrated inFIG. 11-23. Any suitable dielectric material, such as SiO₂ or siliconnitride, TiO₂ or Ta₂O₅, may be used to form the fill-in layer for thediffractive lens and the tiered layer. In the embodiment illustrated inFIG. 11-26, silicon nitride is used to make the fill-in layer and SiO₂is used to make the tiered layer.

The first step in the fabrication process of the tiered SiO₂ layer mayinvolve depositing a hard etch mask 11-2214 directly on top of a SiO₂layer 11-2210 followed by an anti-reflection layer 11-2222 followed by aphotoresist layer 11-2220 by step 11-2201. Any suitable material may beused for the hard etch mask. For example, a-Si may be used for the hardetch mask shown in FIG. 11-26, but other materials are possible. Thenext step may involve applying an ARC and/or a photoresist layer on topof the a-Si hard etch mask. Conventional photolithographic techniquesmay be used to create the variable-period large binary pattern. Thepatterns are transferred to the underlying SiO₂ layer using conventionaletching techniques, such as reactive ion etching techniques, forexample. As shown in FIG. 11-19, SiO₂ layer 11-2210 is etched by step11-2203. SiO₂ layer 11-2210 is striped and cleaned by step 11-2205 usingconventional techniques.

The etch depth of a large diffractive lens step can be any suitabledepth that accomplishes the desired focal length. In the embodimentillustrated in FIG. 11-19, this etch depth into the SiO₂ layer isapproximately 684 nm for the large step. Conventional techniques arethen used to strip the resist and etch mask residues and clean thesurface of the SiO₂ layer. The next step may involve etching the smallsteps on each large step. In the embodiment illustrated in FIG. 11-19,each large steps comprises four smaller steps.

A second Si hard etch mask 11-2244 is then deposited on the patternedSiO₂ layer 11-2310 by step 11-2207. An ARC layer 11-2342 is thendeposited on top of the Si layer 11-2610 followed by a photoresist etchmask layer 11-2640. The second variable-period small binary pattern istransferred to the photoresist and/or the ARC layer by step 11-2209, andSiO₂ layer 11-2210 is etch by to form a Fresnel lens.

In an alternative embodiment, the fabrication steps are similar to thatdescribed in FIG. 11-19, however, two small steps are etched per largestep leaving four steps in total. In other embodiments, any number ofsteps may be used. The small steps are then etched into the SiO₂ layer11-2210. The thickness of a small diffractive lens step can be anysuitable thickness. Additional stages in the fabrication processfollowing creation of the tiered SiO₂ layer 11-2810 by step 11-2801 mayinvolve filling the cavities by step 11-2803 with any suitable highindex lens material 11-2818, such as silicon nitride, for example, anddepositing a transparent layer 11-2828 to create an “embedded Fresnellens”, as illustrated in FIG. 11-20. The tiered structure used for the“embedded Fresnel lens” may have approximately the same and/or smallersize features as the tiered structure used for the offset Fresnel lens.Any method of depositing the silicon nitride may be used such as PECVD,for example. Optionally, the silicon nitride layer may be uniformlypolished down until the top step of the SiO₂ material is exposed by step11-2805. Alternatively, the silicon nitride layer 11-2818 is uniformlypolished but the SiO₂ material is not exposed. In the embodimentillustrated in FIG. 11-21, the tiered structure 11-2910 is formed bystep 11-2901, and filled with a high index material 11-2918 by step11-2903, and polished by step 11-2905. A second layer 11-2928 of SiO₂ isthen deposited via PECVD on top of the polished silicon nitride layer11-2918 and polished via CMP by step 11-2907. In some embodiments, thespacer layer 11-2928 may have a thickness equal to the focal length inthat spacer layer material. Additionally, other suitable transparentspacer layers may be deposited on top of the silicon nitride layer bystep 11-2909. The sample well layer may then be fabricated on top of thetransparent spacer layer and/or additional layers.

Alternatively, in the embodiment illustrated in FIG. 11-22, the tieredlayer 11-3018 for the diffractive lens is made of silicon nitride. Thesilicon nitride layer 11-3018 may be deposited at any suitable thicknesson top of substrate 11-3010 followed by etch mask 11-3014, ARC layer11-3022 and photoresist layer 11-3020. In the embodiment illustrated inFIG. 11-22, the silicon nitride layer is approximately 1 um thick bystep 11-3001. The fabrication processes may be similar to the onedescribed above in regards to creating the tiered, variable-perioddiffractive lens layer in SiO₂. Optionally, a different hard mask may beused to create the silicon nitride tiered layer by step 11-3003. Thesilicon nitride tiered layer may have approximately the same and/orsmaller size features as the SiO₂ tiered layer. After the siliconnitride tiered layer is made, the silicon nitride layer may be coated inany suitable dielectric material 11-3028. In the embodiment illustratedin FIG. 11-30, the silicon nitride layer is coated with transparent(e.g., SiO₂) layer 11-3028 by step 11-3005. The SiO₂ layer may bedeposited using conventional deposition processes such as PECVD, forexample. The SiO₂ layer may then be polished to create a flat, planarsurface. The sample well layer may then be fabricated on top of the SiO₂layer and/or additional layers.

Certain features of the diffractive optic may require a certain degreeof uniformity and/or accuracy during the fabrication process to yield astructure with the desired optical properties. For example, the etchdepth of the large and small steps may require a certain degree ofaccuracy. In some embodiments, an etch depth within 50 or 10% of thetarget may be required to achieve the desired power efficiency into thefocal spot sample well. Additionally, the etching of the lens featuresmay require a certain degree of uniformity. For example, etch uniformitywithin 5% (or 50 nm) is required to achieve the desired focal length.

Any of the lenses described above may be fabricated using any suitabledeposition and etching processes to create improved optical properties.By way of example and not limitation, PECVD may be used. The depositionparameters may be tuned in any suitable way to reduce autoluminescence,reduce lens absorption of luminescence, and/or create a high index ofrefraction. For example, a reduced autoluminescence and lens absorptionmay be achieved by reducing the density of Si—Si bonds, which may formsilicon nanocrystals, during deposition of silicon nitride. In someembodiments, the input gases and their ratios may be modified to reducethe density of Si—Si bonds and silicon nanocrystals. For example, SiH₄and N₂ may be used and their ratios adjusted in any suitable way toreduce the density of Si nanocrystals. In other embodiments, SiH₄ andNH₃ may be used and their ratios adjusted in any suitable way to reducethe density of Si—Si bonds and silicon nanocrystals. For example, theratio of NH₃ to SiH₄ may be at least 10:1. Additionally, tuning thefrequencies that control the plasma during PECVD may be used to improveoptical properties. For example, the low frequency (e.g. below 0.5 MHz)to high frequency (e.g. above 10 MHz) ratio may be at least 1:1.

Additionally, the above described deposition parameters may tune thelens index of refraction to improve optical properties. In someembodiments, the index of refraction for a silicon nitride lens may beless than n=1.92 and associated with a wavelength of 633 nm for asuitable low autoluminescence effect and/or a suitable low absorptionloss. The tuned qualities described above may be related to,proportional, correlated, associated with and/or dependent each other.For example, an index of refraction of n=1.92 is indicative of a lowluminescence and low absorption loss which is related to a low densityof Si—Si bonds and silicon nanocrystals for a lens made of siliconnitride.

Various aspects of the present application may be used alone, incombination, or in a variety of arrangements not specifically discussedin the embodiments described in the foregoing and is therefore notlimited in its application to the details and arrangement of componentsset forth in the foregoing description or illustrated in the drawings.For example, aspects described in one embodiment may be combined in anymanner with aspects described in other embodiments.

Also, the invention may be embodied as a method, of which at least oneexample has been provided. The acts performed as part of the method maybe ordered in any suitable way. Accordingly, embodiments may beconstructed in which acts are performed in an order different thanillustrated, which may include performing some acts simultaneously, eventhough shown as sequential acts in illustrative embodiments.

Use of ordinal terms such as “first,” “second,” “third,” etc., in theclaims to modify a claim element does not by itself connote anypriority, precedence, or order of one claim element over another or thetemporal order in which acts of a method are performed, but are usedmerely as labels to distinguish one claim element having a certain namefrom another element having a same name (but for use of the ordinalterm) to distinguish the claim elements.

Also, the phraseology and terminology used herein is for the purpose ofdescription and should not be regarded as limiting. The use of“including,” “comprising,” or “having,” “containing,” “involving,” andvariations thereof herein, is meant to encompass the items listedthereafter and equivalents thereof as well as additional items.

What is claimed is:
 1. An integrated device comprising: a sample wellarranged on a surface of the device and configured to receive a sample;a waveguide configured to propagate a plurality of pulses of opticalexcitation energy to the sample well; and a sensor, positioned toreceive light from the sample well, that aggregates into at least twotime bins charge carriers of received photons of luminescence emitted bythe sample.
 2. The integrated device of claim 1 wherein the sensoridentifies the sample.
 3. The integrated device of claim 1 wherein thesample is a biological sample.
 4. The integrated device of claim 3wherein the biological sample comprises a target nucleic acid molecule.5. The integrated device of claim 1 wherein the sensor is furtherconfigured to generate a signal indicative of an intensity of theemitted luminescence.
 6. The integrated device of claim 1 furtherincluding a grating coupler, optically coupled to the waveguide.
 7. Theintegrated device of claim 6 further including a first monitoring sensorthat assists in aligning to the grating coupler a beam of opticalexcitation energy through alignment monitoring.
 8. The integrated deviceof claim 7 further including a second monitoring sensor that furtherassists in aligning to the grating coupler the beam of opticalexcitation energy through monitoring power of optical excitation energy.9. The integrated device of claim 1 wherein the sensor includes at leasttwo storage regions that correspond to the at least two time bins.